Anti p src

Cell culture reagents—BK, SU566, ARA-C, DAPI, anti-β-Actin—were purchased from Sigma Aldrich (Merk Millipore). Fetal Bovine Serum (FBS) was from Innoprot (Basque Country). Anti-FGF-2 neutralizing antibody, STAT3 inhibitor VII, PP1, and SU5402 were purchased from Calbiochem (Merk Millipore, Darmstadt, Germany). FGF-2 was from R&D system. Fasitibant was kindly provided by Menarini Ricerche (Florence Italy). Anti-pTYR, anti-FGFR-1, anti-FGFR-2, anti-pFRSα, anti-pERK1/2, anti-ERK1/2, anti-pSTAT3, anti-STAT3, anti-pAKT, anti-AKT, anti-pSRC, and anti-SRC antibodies were from Cell Signaling (Milan, Italy). Anti-FRSα was from R&D system. Anti-FGF-2 was from Merk Millipore (Darmstadt, Germany).

The protein expression was determined using Western blot analysis as described [16 (link)]. Briefly, the kidney tissue and cell lysates were separated on SDS-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. Then, the membranes were incubated with various antibodies including anti-p-eIF2α (1 : 5000, Cell Signaling Technology), anti-p-IRE1α (1 : 4000, Santa Cruz Biotechnology), ATF6α (1 : 5000, Cell Signaling Technology), anti-pJNK (1 : 5000, Cell Signaling Technology), anti-CHOP (1 : 1000, Santa Cruz Biotechnology), anti-pSrc (1 : 1000; Cell Signaling Technology), anti-pFyn (1 : 1000; Santa Cruz Biotechnology), and anti-β-actin (1 : 1000) overnight at 4°C on a shaker. Then, the membranes were incubated with respective secondary antibodies, washed, and reacted with an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The bands were quantified by using ImageJ software and normalized by β-actin.

3, 3′-dithiobis sulfosuccinimidylpropionate (DTSSP) and beta-D-Lactose were purchased from Thermo Scientific (Pittsburgh, PA). Sucrose was purchased from MP Biomedicals (Solon, OH). Ouabain octahydrate, cis-diammineplatinum dichloride (CDDP), doxorubicin hydrochloride (DXR) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Pyrazolo pyrimidine (PP2), a Src kinase inhibitor, was purchased from Tocris Bioscience (Bristol, UK).
Customized polyclonal rabbit anti-Gal-3 antibody was created by Pierce Biotechnology; mouse anti-V5 and purified mouse IgG were purchased from Invitrogen; polyclonal goat anti-Na⁺/K⁺-ATPase alpha1 and monoclonal mouse anti-Mdr-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal rabbit anti-Mdr was purchased from Oncogene Research Products (Cambridge, UK); mouse anti-beta-actin was purchased from Sigma-Aldrich; rabbit anti-Src and anti-p-Src were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA); monoclonal mouse anti-phosphoserine was purchased from abcam (Cambridge, MA).

Cells were harvested and lysed in SDS lysis buffer (PMSF, Protease, and Phosphatase Inhibitor Cocktail added) for 30 min at 4°C. Total protein was extracted from tissues with T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The proteins were dissociated and separated by SDS/PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies. The primary antibodies used for western blotting and their sources were as follows: anti-SEMA3C (Proteintech, 19242-1-AP), anti-GAPDH (Proteintech, 60004-1-Ig), anti-p-ERK1/2 (Cell Signal Technology, #4370), anti-ERK1/2 (Cell Signal Technology, #4695), anti-p-EGFR (Cell Signal Technology, #3777), anti-EGFR (Proteintech, 18986-1-AP), anti-p-Her2 (Cell Signal Technology, #2244), anti-Her2 (Cell Signal Technology, #2244), anti-Her2 (Proteintech, 18299-1-AP), anti-p-MET (Cell Signal Technology, #3077), anti-MET (Proteintech, 25869-1-AP), anti-p-SRC (Cell Signal Technology, #6943), and anti-SRC (Proteintech, 11097-1-AP). Antigen-antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology #7074; #7076) with enhanced chemiluminescence (ECL) western blot detection reagent (Merck Millipore) according to the manufacturer's protocol.

For protein extraction, the cells directly harvested with 2X lysis buffer (Cell Signaling Technology, 9803), 100X phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, 78,830), 10X PhosSTOP, and cOmplete Protease Inhibitor cocktail (Roche). The cell lysates (30 µg protein per sample) were separated by electrophoresis, transferred to PVDF membranes (Millipore, IPVH00010), and were incubated with the antibodies. The anti-Src, anti-pSrc, anti-STAT3, anti-pSTAT3, and anti-EEA1 antibodies (1:1000) were purchased from Cell Signaling Technology, while the anti-FGFR4, anti-LaminB1 (1:200), and anti-beta-actin (1:1000) antibodies were purchased from Santa Cruz Biotechnology. The anti-FGF19 (1:500) antibody was procured from R&D Systems, while the anti-phosphotyrosine antibody, clone 4G10 (1:1000) was procured from Millipore. The secondary antibodies used (1:5000) were HRP-conjugated mouse anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, and bovine-anti goat IgG-HRP-linked antibodies (Santa Cruz Biotechnology). Detection was performed using an ECL™ Prime Western Blot System (GE Healthcare) and an Amersham Imager 600 (GE Healthcare). The bands were quantified with ImageJ, a Java-based image analysis package that is widely used for measuring density.

NaHS was administered instead of H₂S. NaHS and the Pyk2 inhibitor (PF431396) were obtained from Sigma-Aldrich; The FAK specific inhibitor (PF573228), the Src inhibitor (PP2), and the Rac1 inhibitor (NSC23766) were purchased from Selleck Chemicals. CES-siRNA (sc-142618) was obtained from Santa Cruz. Rhodamine was obtain from Cytoskeleton, and DAPI from Beyotime; Following antibodies were used: anti-Rac, anti-Fak, anti-p-FAK³⁹⁷, anti-p-FAK⁹²⁵, anti-Src, anti-p-Src, anti-β-actin, anti-Integrin β1, anti-Integrin β3, anti-Cavenolin-1,anti-p-Pyk2 (Cell Signaling); anti-CSE (Santa Cruz); anti-Integrin β1-FITC, anti-Galectin-3 (eBbioscience); anti-CD68 (Biolegend); Lactate dehydrogenase (LDH) assay from BeyotimeInstitute of Biotechnology.