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Results: 5

1.
Figure 1

Figure 1. From: Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

SOCS1 has the highest affinity for oncogenic Y505FLck kinase. (A) COS7 cells were cotransfected with 5 μg of pcDNA/Y505FLck and 5 μg of pEBG (empty vector), pEBG/SOCS1, pEBG/SOCS2, pEBG/SOCS3 or pEBG/CIS. A small aliquot of normalized whole cell lysate was analyzed by immunoblotting with anti-Myc antibody to detect Y505FLck (top panel). Following glutathione pulldown, anti-Myc and anti-GST immunoblotting was performed to detect Y505FLck (middle panel) and GST or GST-SOCS fusion proteins (bottom panel), respectively. (B) The relative affinity of SOCS1, SOCS2, SOCS3 and CIS for Y505FLck was calculated as described in “Materials and methods”. The affinity of GST for Y505FLck was set as 1 and values represent the means ± S.D. (n=3; *, p<0.05; ***, p<0.001).

SRIVIDYA VENKITACHALAM, et al. Oncol Rep. ;25(3):677-683.
2.
Figure 2

Figure 2. From: Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

SOCS1 interacts with the oncogenic Y505FLck kinase. (A) COS7 cells were cotransfected with 5 μg of pcDNA/Y505FLck (+) or pcDNA (−) and 5 μg of pEBG/SOCS1 (+) or pEBG (−). A small aliquot of normalized whole cell lysate was analyzed by anti-GST immunoblotting to detect GST and GST-SOCS1 (top panel). Following Lck immunoprecipitation, western blot was performed with anti-GST antibody to detect GST and GST-SOCS1 (middle panel) and anti-Lck antibody to detect Y505FLck (bottom panel). The position of immunoprecipitating antibody light chain (LC) is also indicated. (B) COS7 cells were cotransfected with SOCS1 and Y505FLck expression constructs. Immunofluorescence microscopy was performed as described in “Materials and methods” to visualize SOCS1 (red), Y505FLck (green), and DAPI-stained nuclei (blue). In the three-color merge image (bottom panel), arrows point to regions of interaction between SOCS1 and Y505FLck (yellow). Original magnification, x60.

SRIVIDYA VENKITACHALAM, et al. Oncol Rep. ;25(3):677-683.
3.
Figure 4

Figure 4. From: Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

Kinase domain of Lck is sufficient to mediate interaction with SOCS1. (A) Schematic diagrams of Lck functional domains and truncation mutants. (B) COS7 cells were cotransfected with 5 μg of pEBG or pEBG/SOCS1 and 5 μg of pcDNA with various Lck mutant constructs. A small aliquot of normalized whole cell lysate was analyzed by anti-Myc immunoblotting to detect Myc-tagged mutant Lck (top panel). A strong non-specific band was detected below the truncated Lck proteins (top panel). Following glutathione pulldown, western blot was performed with anti-Myc antibody to detect mutant Lck (middle panel) and anti-GST antibody to detect GST and GST-SOCS1 (bottom panel). (C) The relative affinity of mutant Lck for GST-SOCS1 was calculated as described in “Materials and methods”. The affinity of N32Lck for GST- SOCS1 was set as 1 and values represent the means ± S.D. (n=2; *, p<0.05).

SRIVIDYA VENKITACHALAM, et al. Oncol Rep. ;25(3):677-683.
4.
Figure 3

Figure 3. From: Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

Phosphorylated Tyr394 in the Lck kinase domain is important in SOCS1 association. (A) Schematic diagrams of Lck functional domains and point mutations. (B) COS7 cells were cotransfected with 5 μg of pEBG or pEBG/SOCS1 and 5 μg of pcDNA without or with various Lck mutant constructs. A small aliquot of normalized whole cell lysate was analyzed by anti-Myc immunoblotting to detect Myc-tagged mutant Lck (top panel). Following glutathione pulldown, western blot was performed with anti-Myc antibody to detect mutant Lck (middle panel) and anti-GST antibody to detect GST and GST-SOCS1 (bottom panel). (C) The relative affinity of mutant Lck for GST-SOCS1 was calculated as described in “Materials and methods”.
The affinity of Y505FLck for GST-SOCS1 was set as 1 and values represent the means ± S.D. (n=3; ***, p<0.001).

SRIVIDYA VENKITACHALAM, et al. Oncol Rep. ;25(3):677-683.
5.
Figure 5

Figure 5. From: Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

A functional SH2 domain in SOCS1 is involved in binding to the oncogenic Lck kinase. (A) Schematic diagrams of SOCS1 functional domains and point mutations. KIR and ESS represent the kinase inhibitory region and the extended SH2 subdomain, respectively. A highly conserved SOCS box in the carboxy-terminus is also illustrated. (B) COS7 cells were cotransfected with 5 μg of pcDNA/Y505FLck and 5 μg of pEBG without or with wild-type (WT) and mutant SOCS1 constructs. A small aliquot of normalized whole cell lysate was analyzed by anti-Lck immunoblotting to detect Y505FLck (top panel). Following glutathione pulldown, western blot was performed with anti-Lck antibody to detect Y505FLck (middle panel) and anti-GST antibody to detect GST and GST-SOCS1 (bottom panel). (C) The relative affinity of wild-type and mutant SOCS1 for Y505FLck was calculated as described in “Materials and methods”. The affinity of wild-type GST-SOCS1 for Y505FLck was set as 1 and values represent the means ± S.D. (n=3; **, p<0.01).

SRIVIDYA VENKITACHALAM, et al. Oncol Rep. ;25(3):677-683.

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