Anti gapdh 14c10

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.

Antibodies purchased from Cell Signaling Technology were used at a dilution of 1:1000 and include phospho‐S6 ribosomal protein (S235/236) (D57.2.2E) XP (R) (rabbit monoclonal, #4858), phospho‐AKT (Ser 473) (D9E) XP(R) (rabbit mAb, #4060), phospho‐p44/42 MAPK (T202/Y204) (rabbit, #9101), total AKT (rabbit, #9272), total MAPK (mouse, #9107), total S6 (rabbit, #2217), Mcl‐1 (rabbit, #5453), Bcl‐xL (rabbit, #2764), Bcl‐2 (human specific, #2872) and beta‐tubulin (rabbit polyclonal, #2146). Anti‐GAPDH (14C10) (rabbit mAb, #2118) (Cell Signaling Technology) was used at a dilution of 1:3000.

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.