Anti rac1b

Our Western blotting procedure was described in detail earlier [16 (link),17 (link),43 (link)]. Total protein concentrations were determined with the DC Protein Assay (BioRad). Proteins were fractionated by PAGE on mini-PROTEAN TGX any-kD precast gels and blotted onto PVDF membranes. The primary antibodies included anti-HSP90 (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-13119), anti-Rac1b (Merck Millipore, Darmstadt, Germany, #09-271), anti-E-cadherin and anti-Cip1/WAF1 (BD Transduction Laboratories, Heidelberg, Germany, #610181 and #610233, respectively), anti-Snail and anti-phospho-ERK1/2 (Cell Signaling Technology, Frankfurt/Main, Germany, #4719 and #4370, respectively), anti-ERK1/2 (R&D Systems, Wiesbaden, Germany, #AF1576), anti-GAPDH (14C10, Cell Signaling Technology, #2118), and anti-β-actin (Sigma). Incubation with HRP-linked secondary antibodies (Cell Signaling Technology, anti-rabbit, #7074, and anti-mouse, #7076) was followed by chemoluminescent detection of proteins on a ChemiDoc XRS+ System with Image Lab Software (BioRad) using Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to bands for the housekeeping genes GAPDH or HSP90.

The following primary antibodies were used: Anti-phospho-p38 (#9211), anti-p38 (#9212), anti-phospho-ERK1/2 (#4370), anti-E-Cadherin (#3195), anti-Snail (#4719) (all from Cell Signaling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-MKK6 (sc-6073), anti-TGF-β receptor I/ALK5 (V22, #sc-398), anti-TGF-β1 (3C11, #sc-130348) (all from Santa Cruz Biotechnology, Heidelberg, Germany), anti-ERK1/2 (#AF1576, R&D Systems, Wiesbaden, Germany) anti-Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), anti-Rac1 (#610650), anti-Cip1/WAF1 (#610233) (both from BD Biosciences, Heidelberg, Germany), anti-β-actin (#A1978, Sigma, Deisenhofen, Germany). anti-Flag M2 (F3165, Sigma), HRP-linked anti-rabbit (#7074), anti-mouse (#7076) and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology, anti-goat secondary antibody (#ab6741) was from Abcam (Cambridge, UK). Recombinant human (rh) TGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/ml. The p38 inhibitor SB203580, the MEK1 inhibitor U0126 and the ALK5 inhibitor SB431542 were purchased from Calbiochem and used at a concentration of 10 μM (SB203580, UO126) and 5 μM (SB431542). Treatment of cells with these inhibitors for up to 48 h had no gross effect on cell viability.

The following primary antibodies were used: anti-Smad3, #ab40854, Abcam (Cambridge, UK), anti-Smad2, #sc-7947, anti-HSP90, #sc-13119, and anti-TGF-β receptor I, V22, #sc-398, Santa Cruz Biotechnology (Heidelberg, Germany), anti-RAC1B, #09-271, Merck Millipore (Darmstadt, Germany), and anti-phospho-Smad3(Ser423/425), #9514, and anti-GAPDH (14C10), #2118, Cell Signaling Technology (Frankfurt am Main, Germany). HRP-linked anti-rabbit, #7074, and anti-mouse, #7076, secondary antibodies were from Cell Signaling Technology. Recombinant human TGF-β1, #300-023, was provided by ReliaTech (Wolfenbüttel, Germany), and SIS3 from Merck/Calbiochem. The C-terminal phosphorylation-resistant SMAD3 mutant (SMAD3-mut3A), in which the three serine residues at position 422, 423, and 425 were replaced by alanines, was constructed by amplifying the entire coding sequence of SMAD3 with forward primer 5’-ACCATGGCGTCCATCCTGCCTTTC-3’ (Kozak consensus sequence underlined) and reverse primer 5’-TCTAAGCCACAGCGGCACAGCGGATGCTTGGGG-3’ (mutant nucleotides underlined) with Pfu polymerase and subsequent insertion in pcDNA3 digested with EcoRV. The desired sequence changes were confirmed by sequencing.