siRNA knockdown of p38α inhibits translocation of FGF1. (A to C) NIH 3T3 cells were transfected with control siRNA (ctrl) or three different siRNAs specific for mouse p38α (α1, α2, and α3). (A) Total cell lysates were analyzed for p38α (i) and total p38 MAPK (ii) by Western blotting. In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells. Phosphorylated FGF1 (33P-FGF1) was detected by fluorography (iii), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [iv]). (B) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the presence of 10 μM anisomycin. (C) In vivo phosphorylation of FGF1 was analyzed in the siRNA-transfected cells in the absence (i) or presence (ii to v) of LMB, and the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions before extraction of FGF1. (D) Nuclear and cytoplasmic fractions of NIH 3T3 cells obtained by the cellular fractionation method described for panel C were tested for the presence of the cytoplasmic proteins rab5a, calreticulin, and ERK1/2 and the nuclear protein lamin A by specific antibodies. (E and F) BJ cells were mock transfected or transfected with control siRNA (ctrl), two different siRNAs specific for human p38α (α1 and α2), or two different siRNAs specific for human p38β (β1 and β2). (E) The transfected cells were lysed and tested for the amount of p38α (i), p38β (ii), and ERK1/2 (loading control, [iii]) by immunoblotting. Translocation of FGF1 was tested in the transfected cells in an in vivo FGF1 phosphorylation assay. Phosphorylated FGF1 (33P-FGF1) was detected by fluorography (iv), and the total cellular uptake was detected by anti-FGF1 immunodetection (total FGF1 [v]). No FGF1 was added in the first lane, panels iv and v. (F) siRNA-transfected BJ cells were incubated with in vitro-labeled 35S-FGF1 and heparin for 6 h, and then the cells were fractionated into membrane (M), cytosolic (C), and nuclear (N) fractions. FGF1 was extracted from each fraction by binding to heparin-Sepharose and analyzed by SDS-PAGE and fluorography. (C and N fractions were exposed to film four times longer than the M fractions.) (G) The subcellular fractions obtained by fractionation as described for panel F were tested for the presence of the membrane-associated proteins Rab5a and calreticulin, the cytosolic protein ERK1/2, and the nuclear protein lamin A by specific antibodies.