Sc 514302

Tissue samples of the dorsal region of the spinal cord (L4–5, semi-sectioning along the longitudinal-horizontal direction) were removed. Equal amounts of the protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF). Primary antibodies against the following proteins were used: brain-derived neurotrophic factor (BDNF, 1:100, SC-20981, Santa Cruz), c-fos (1:500, SC-52, Santa Cruz), growth-associated protein 43 (GAP43, 1:500, ab121217, Abcam), p-ERK (1:500, SC-7383, Santa Cruz), ERK 1/2 (1:500, SC-514302, Santa Cruz), transient receptor potential vanilloid 1 (TRPV1, 1:500, ab203103, Abcam), and inducible nitric oxide synthase (i-NOS, 1:500, ab3523, Abcam). Target protein expression was normalized to β-actin (1:2000, 8457S, Cell Signaling Technology) expression. The quantification of band intensity was carried out using Image J (National Institutes of Health, USA).

Radioimmunoprecipitation assay buffer (Biosesang, Seoul, Republic of Korea), containing protease (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase (PhosSTOP; Roche, Basel, Switzerland) inhibitor cocktails, was used to isolate total cellular proteins. Protein concentrations are measured by BCA assay. Western blotting was conducted using standard protocols. 5% skim milk was used for blocking the nonspecific binding for 1 h. Primary antibodies were mouse anti- extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) antibody (sc514302, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-pERK1/2 antibody (sc136521, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-YAP antibody (sc-376830, Santa Cruz, CA, USA, 1:500 dilution), mouse anti-pYAP antibody (PA5-17481, Invitrogen, 1:500 dilution) or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1:5000 dilution). A horseradish peroxidase (HRP) conjugated secondary antibody and a WEST-Queen™ Western Blot Detection Kit (iNtRON biotechnology, Seongnam, Republic of Korea) were used to detect immunoreactive bands. Data were quantified by video image analysis (Luminograph II, Atto, Tokyo). Protein bands were measured by Image J.

Western blotting was performed as previously described [5 (link)]. In brief, rats were anesthetized with isoflurane at the established time points after ICH and transcardially perfused with cold PBS. The right brain hemisphere was homogenized in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology, USA) and further centrifuged at 14000 rpm for 30 min. Equal amounts of protein (4 μL, 30 μg) were loaded onto a 10% SDS-PAGE gel. After separation, the proteins were transferred to a nitrocellulose membrane, which was further blocked for 2 h with a blocking solution. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-albumin (1 : 500, ab207327, Abcam, USA), anti-p-ERK (1 : 1000, Santa Cruz Biotechnology, USA), anti-ERK (1 : 1000, sc-514302, Santa Cruz Biotechnology, USA), anti-Nrf2 (1 : 1000, GTX103322, Gene Tex), anti–HO-1 (1 : 1000, ab68477, Abcam, MA, USA), anti-Romo1 (1 : 1000, Aviva Systems Biology, San Diego, CA), anti-Bcl2 (1 : 1000, ab59348, Abcam, MA, USA), and anti-Bax (1 : 1000, Littleton, CO). The membranes were incubated with an anti-β-actin antibody (1 : 5000, sc-47778, Santa Cruz Biotechnology, TX, USA).) The results were normalized using β-actin as a loading control. The relative density of the blot bands was quantified by densitometry using the ImageJ software.

An RT-qPCR and Western blot analysis were performed as reported previously [8 (link),10 (link)]. mRNA and protein samples were prepared 48 h after seeding, adenovirus transduction, or irradiation. The primers used for the RT-qPCR are summarized in Table S1. β-Actin was used as an internal control for calculating the relative mRNA expression levels. Antibodies against 75-kDa glucose-regulated protein (GRP75; sc-133137), extracellular signal-regulated kinases (ERK) 1/2 (sc-514302), GABARAP (sc-377300), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062), parkin (sc-32282), PINK1 (sc-518052), SOD1 (sc-101523), and SOD2 (sc-133134) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunoblotting was carried out as specified [44 (link)] using Spry4 antibodies raised to the N-terminal 60 aa of the protein, and purified as described [42 (link)]. Antibodies recognizing phosphorylated extracellular signal-regulated kinase (pERK) (#9101 were purchased from Cell Signaling Technology (Danvers, MA, USA) and diluted 1:1000. Furthermore, from Santa Cruz (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) purchased antibodies directed against ERK 1/2 (sc-514302) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-365062) were used in a 1:500 and 1:20,000 dilution, respectively. The HRP-coupled secondary antibodies were purchased from GE Healthcare (Chalfont St. Giles, UK) and usually incubated in a 1:5000 dilution.

The cultured pancreatic cancer cells were harvested and lysed with RIPA lysis buffer containing cOmplete protease inhibitor cocktail (04693132001; Roche, Switzerland) and PhosSTOP phosphatase inhibitor (04906845001; Roche, Switzerland). The protein concentration was determined with a BCA protein quantification assay. Proteins in each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected on PVDF membranes. The antibodies used for Western blot were as follows: anti-ENO1 (ab227978, 1:1,000; Abcam, USA), anti-caspase-3 (9,662, 1:1,000; CST, China), anti-GAPDH (sc-47724, 1:3,000; Santa Cruz, USA), anti-CA9 (ab243660, 1:1,000; Abcam, USA), anti-ERK (sc-514302, 1:2,000; Santa Cruz, USA), and anti-phospho-Erk1/2 (Thr-202/Tyr-204) (4,370, 1:1,000; CST, China). Blots of the target proteins were detected with WesternBright ECL HRP substrate (R-03031-D2; Advansta, USA) and visualized with a C-DiGit Blot Scanner (LI-COR Biosciences, USA).