Anti gapdh mab

The human renal cell carcinoma cell lines, 786-O and ACHN, were purchased from the Shanghai Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China) and cultured in DMEM and RPMI-1640 Medium, containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, at a 37°C incubator with 5% CO₂. The murine renal adenocarcinoma cell line Renca was purchased from Cobioer Biosciences and cultured in RPMI-1640 supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1 mM NEAA, and 1 mM sodium pyruvate solution. Most of the experiments were carried out when cells were grown to 70% confluence. Dulbecco’s Modified Eagle Medium and RPMI-1640 Medium were purchased from Gibco. DMSO was purchased from SIGMA. PFD was from Selleckchem (#S2907). Recombinant human TGF-β1 (#100-21, Peprotech) was reconstituted in 4 mM HCl containing 1 mg/ml bovine serum albumin (BSA). Anti-E-cadherin mAb (#AB40860, Absci), anti-N-cadherin mAb (#AB21474, Absci, China), anti-c-Ski mAb (ab131134, Abcam), anti-Smad2/3 mAb (#5678, CST), anti-phosphorylated Smad2 mAb (#8828, CST) and anti-GAPDH mAb (#sc-32233, Santa Cruz) were used for Western blot. PE/Cy7 anti-mouse CD45 Antibody (#147704, Biolegend), APC anti-mouse/human CD11b Antibody (#101212, Biolegend) and FITC anti-mouse Gr1 Antibody (#108405, Biolegend) were used for flow cytometry.

For staining for AJ proteins, the following monoclonal antibodies (mAbs) were used: anti-β-catenin (CAT-5H10, mouse IgG, Thermo Fisher Scientific, Waltham, MA, USA) and anti-phospho β-catenin (S33/S37/T41) (Rabbit, Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies used were goat anti-mouse IgG conjugated to Alexa Fluor 647 (Abcam, Cambridge, UK) and goat-anti-rabbit IgG conjugated to Alexa 568 (Abcam). The same panel of mAbs was used for Western blotting along with the anti-GAPDH mAb (Rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA). For chemical inhibition of the GSK-3β activity, we used a cell-permeable GSK-3 peptide inhibitor (Millipore Sigma™ Calbiochem™, Steinheim, Germany) at concentrations (0.2–40 μM) that do not affect the cell viability, as previously demonstrated [41 (link)].

Immunoblotting was performed as previously described in detail [17 (link)]. Briefly, cells were lysed with RIPA lysis buffer (Nacalai Tesque) containing 1 mM PMSF, 0.15 U/ml aprotinin, 10 mM EDTA, 10 ng/ml sodium fluoride and 2 mM sodium orthovanadate. Cellular proteins were quantified using a DC Protein Assay (Bio-Rad, Richmond, CA). Equal amounts of proteins were loaded onto the gels, separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). The membranes were probed with primary antibodies (Abs) such as anti-microtubule- associated protein 1 light chain 3 (LC3) B antibody (Ab) (NB600-1384; Novus Biologicals, Inc., Littleton, CO), and anti-phosoho-eIF2α (Ser51) Ab (#9721S), anti-CHOP (GADD153) monoclonal (m) Ab (#2895S), anti-p70S6K Ab (#9202S), anti-phospho-p70S6K (Thr389) Ab (#9205S), anti-PARP Ab (#9542) (Cell Signaling Technology, Danvers, MA, USA), and anti-p62 (sequestosome-1) mAb (sc-28359), anti-GAPDH mAb (sc-32233), and anti-β-actin mAb (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-XBP-1s Ab (S647501) (BioLegend, San Diego, CA). Immunoreactive proteins were detected with horseradish peroxidase-conjugated second Abs (Jackson, West Grove, PA) and an enhanced chemiluminescence reagent (ECL) (Millipore). Densitometry was performed using a Molecular Imager, ChemiDoc XRS system (Bio-Rad).