WT or P125H, Y86A, Y106A, and Y159A mutations in the TIR domain of Mal WT or P125H, Y86A, Y106A, Y159A, Y187A, Y195A and Y196A Flag-Mal variants were overexpressed in HEK293T cells (the vector input was 10 µg per 100-mm dish), and pCDNA3 transfection was used as a negative control. Cell extracts were analyzed by immunoblotting with α-Flag Ab (A and B) or Abs against IκB-α, p-p38, and total p38. β-Actin immunoblot was used to control for protein loading (A). (A, top two panels): Mal species were immunoprecipitated with α-Flag Ab and subjected to immunoblot analyses with α-p-tyrosine Ab PY20 or α-Flag Ab to detect Mal phosphorylation and total protein expression, respectively. (B): NF-κB reporter activation was analyzed in cell extracts by measuring luciferase vs. β-galactosidase activities, and the total levels of Mal were analyzed in the same cell lysates by immunoblotting with α-Flag Ab (bottom panel). (C): RNA was isolated, cDNA was prepared and IL-8 and HPRT mRNA expression levels were analyzed by real-time RT-PCR with gene-specific primers. *p<0.005 reflect statistically significant differences in gene expression and reporter activation between Flag-Mal WT and the indicated mutants. Shown are the results of a representative (n=3) experiments.

(A): WT, P125H, Y86A, Y106A, Y159A, and Y196A Flag-Mal variants were co-expressed in HEK293/TLR4/MD-2 cells along with Btk (the input vector amounts were 4 µg each per 100-mm dish). After recovery for 24 h, cells were treated with medium or stimulated for 30 min with 100 ng/ml LPS, and cell lyzates were prepared. Mal species were immunoprecipitated with α-Flag Ab, and their interactions with Btk were analyzed by immunoblotting with α-Btk Ab and compared to total protein levels of Mal, as detected by Western blot analyses of Flag-Mal immunoprecipitates with α- Flag-HRP Ab. Total protein levels of overexpressed Btk were analyzed by immunoblotting of cell lysates with α-Btk Ab. (B): Human monocytes were pretreated for 20 h with medium or tolerized with 10 ng/ml LPS, washed and re-stimulated with 100 ng/ml LPS for the indicated time intervals. Total endogenous Mal proteins were immunoprecipitated from cell extracts with α-Mal Ab, and subjected to immunoblot analyses with α-Btk and α-Mal Abs to assess Mal-Btk associations and Mal total protein levels, respectively. The results of a representative experiment (n=3) are shown.

pEFBOS expression vectors encoding Flag-tagged WT, P125H, Y86A, Y106A, Y159A, or Y196A Mal variants (5 µg per dish) were co-transfected into HEK293T cells along with pCDNA3-AU-1-MyD88 (A and B) or pCDNA3-YFP-MyD88 (C) (5 µg per dish each) in 100-mm tissue culture dishes. Cell extracts were prepared, Flag-Mal species or AU-1-MyD88 (A, B) and YFP-MyD88 (C) were immunoprecpitated with the corresponding α-epitope Abs. AU-1-MyD88 and YFP-MyD88 immunoprecipitates were analyzed for interactions with Flag-Mal variants by immunoblotting with α-Flag Ab, and total protein levels of overexpressed MyD88 were measured with α-AU-1 or α-GFP Abs. Flag-Mal immune complexes were immunoblotted with α-AU-1 (A and B) or α-GFP (C) Abs to examine associations with epitope-tagged MyD88 vs. total Mal expression analyzed by immunoblotting with α-Flag-HRP. (D) pCDNA3-AU-1-MyD88 (0.1 µg per well) and pEFBOS-Flag-Mal expression plasmids encoding WT or mutant Mal variants (0.5 µg per well) were transfected in HEK293T cells along with pELAM-luciferase (0.3 µg per well) and pTK-Renilla luciferase (0.1 µg per well) reporters. NF-κB reporter activation was studied by measuring firefly vs. Renilla luciferase activities. The data of a representative experiment (n=4) are shown.

(A) HEK293T cells were plated into 100-mm tissue culture dishes and transiently transfected with pCDNA3-CD14 (1 µg per dish), pCDNA3-TLR4 (4 µg per dish), pEFBOS-HA-MD-2 (1 µg per dish), and pEFBOS-Flag-Mal (WT, 4 µg per dish). To study LPS-inducible Mal tyrosine phosphorylation, the 293/TLR4/MD-2 stable cell line (B) and human monocytes (C) were used. Cells were treated with medium or stimulated with 100 ng/ml LPS as shown. Flag-Mal or endogenous Mal were immunoprecipitated with α-Flag or α-Mal Abs, respectively, and subjected to immunoblotting with α-p-tyrosine Ab PY20 (Mal phosphorylation) and α-Flag (A) or α-Mal (B and C, top panels) Abs (total protein expression of overexpressed or endogenous Mal). Alternatively, total tyrosine-phosphorylated proteins were immunoprecipitated with PY20 Ab, followed by immunoblot detection of p-Mal within this fraction, using Abs against endogenous Mal (B and C, middle panels). To examine total levels of endogenous Mal proteins, Mal proteins were immunoprecipitated with Ab against endogenous Mal and subjected to immunoblotting with α-Mal Ab (B and C, bottom panels). Results of a representative experiment (n=3) are shown.

The HEK293/TLR4/MD-2 stable cell line (A) or human monocytes (B) were pretreated for 20 h with medium or 10 ng/ml LPS, washed and re-stimulated with 100 ng/ml LPS as indicated. Endogenous Mal proteins were immunoprecipitated from cell extracts with α-Mal Ab, followed by immunoblotting with α-p-tyrosine Ab PY20 (to measure Mal phosphorylation) and α-Mal Ab (to detect Mal total protein expression). For controlling cell activation and endotoxin tolerance induction, IRAK-1 and p38 phosphorylation were examined by immunoblotting of whole cell lysates with the corresponding Abs. The results of a representative experiment are shown, and densitometric quantification of relative protein levels of p-Mal, p-p38, and p-IRAK-1 (normalized to total Mal, p38, unmodified IRAK-1 and β-actin, respectively) from three (A) and six (B) experiments are presented on the left.

(A) WT, P125, Y86A, Y106A, Y159A, and Y196A Flag-Mal variants were co-expressed in HEK293T cells along with CD4-TLR4 (5 µg of each expression vector per 100-mm dish), and pCDNA3 transient transfection was used as a negative control. CD4-TLR4 and Flag-Mal proteins were immunoprecipitated with α-CD4 and α-Mal (to avoid co-precipitation of CD4-TLR4 that also expresses Flag tag) Abs, respectively. Flag-Mal proteins (~35 kDa) associated with TLR4 were detected by immunoblotting of CD4-TLR4 immunoprecipitates with α-Flag Ab, and compared to total expression of CD4-TLR4, as detected by the immunoblotting with α-CD4 Ab. Reciprocally, Mal proteins were immunoprecipitated with α-Mal Ab, and Mal interactions with CD4-TLR4 vs. total expression of transfected Flag-Mal were analyzed by immunoblotting with α-Flag Ab to detect CD4-TLR4 (~75 kDa) and Flag-Mal (~35 kDa), respectively. Total expression levels of transfected Flag-Mal and CD4-TLR4 were also examined by immunoblotting of whole cell lysates with α-Flag and α-CD4 Abs, respectively. (B): P125H, Y86A, and Y196A Mal species or pCDNA3 were expressed in 293/YFP-TLR4/MD-2 cells, cells were recovered for 20 h and stimulated for 5 min with 100 ng/ml LPS. Thereafter, p38 phosphorylation was examined by immunoblotting of cell lysates with α-p-p38 Ab, and YFP-TLR4 and Flag-Mal protein levels were assessed by immunoblotting with α-GFP and α-Flag Abs, respectively. β-actin immunoblot was run to control for equal protein loading. (C). pEFBOS-Flag-Mal expression vectors encoding P125H, Y86A, Y106A, and Y159A Mal species were co-transfected in 293/TLR4/MD-2 cells with pELAM-lucifearse and pCMV-β-galactosidase reporters. Cells were recovered for 20 h, stimulated with 1µg/ml LPS for 6 h, and luciferase vs. β-galactosidase activities were measured in cell extracts. *p<0.05 reflect statistically significant differences in reporter activation between pCDNA3 control and the indicated mutants. Results of a representative experiment (n=3) are depicted.

Flag-tagged (A) or HA-tagged (B) WT, P125H, Y86A or Y196A Mal variants were co-expressed in HEK293T cells along with Myc-IRAK-2 (A and C) or Flag-TRAF-6 (B and D) similar to the protocol described in Fig. 6. Mal species were immunoprecpitated with α-Flag (A) or α-HA (B) Abs, and analyzed for interactions with Myc-IRAK-2 (A) or Flag-TRAF-6 with α-Myc or α-Flag Abs. In addition, Myc-IRAK-2 (A) and Flag-TRAF-6 (B) were immunoprecipitated with α-Myc or α-Flag Abs, and immunoblotted with α-Flag (A) or α-HA Abs to examine the presence of co-immunoprecipitated Mal species. Total protein levels of all expressed protein were analyzed with the corresponding α-epitope Abs. (C and D): WT, Y86A, Y106A, and Y196A Flag-Mal (C) or HA-Mal (D) species were co-expressed together with Myc-IRAK-2 (C) or Flag-TRAF-6 (D). For reporter assays, expression vectors encoding IRAK-2 and TRAF-6 were used at input amounts of 50 ng per well that gave ~30–40% of plateau levels of NF-κB reporter activation caused by overexpression of these intermediates. pEFBOS-Mal plasmids were used at 500 ng per well, and the NF-κB and Renilla luciferase reporters were used as described in Fig. 6. Firefly vs. Renilla luciferase activities were measured in cell lysates to assess NF-κB reporter activation. The data of a representative experiment (n=3) are presented.