Horseradish peroxidase conjugated donkey anti rabbit igg

Verteporfin (#SML0534) and antibodies recognizing β-actin (#a2228), FLAG^® M2 (#F1804), glyceraldeyde-3-phosphate dehydrogenase (GAPDH, #CB1001) and 17-β-estradiol (#E1024) were purchased from Merck (Billerica, MA, USA). Antibodies recognizing YAP/TAZ (#8418), p-YAP (Ser127) (#4911), zinc finger E-box binding homeobox1 (ZEB1, #3396), Src (#2108), p-Src (Tyr416) (#2101), horseradish peroxidase-conjugated donkey anti-rabbit IgG (#7074), and horseradish peroxidase-conjugated donkey anti-mouse IgG (#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies recognizing E-cadherin (#610181) and N-cadherin (#610920) were purchased from BD Biosciences (San Jose, CA, USA). The antibody recognizing p-YAP (Tyr357) (ab62751) was purchased from Abcam (Cambridge, United Kingdom). Antibodies recognizing Vimentin (sc-32322) was purchased from Santa Cruz biotechnology (Dallas, TX, USA). V5 Tag (#A190-120A), Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG were purchased from Thermo Fisher Scientific (Cleveland, OH, USA). Saracatinib (#S1006) and PP2 (#S7008) were purchased from Selleckchem (Houston, TX, USA). G418 was purchased from Biosesang (Gyeonggi-do, South Korea).

Yeast cells cultured in the inducible (SG + Ade + Leu + His) medium were lysed by vortexing with acid-washed glass beads of 0.45–0.55 diameter (Sigma Aldrich, USA) in sodium dodecyl sulfate (SDS)-sample buffer and subjected to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis using the rabbit anti-Flag (DYKDDDDK tag) primary antibody (Cell Signaling, USA) and horseradish peroxidase conjugated donkey anti-rabbit IgG as the secondary antibody (Cell Signaling, USA). Protein signals were detected by Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, USA).

The spinal cord was rapidly removed by hydro-extrusion under inhalational anesthesia with sevoflurane (N = 7 per group). Lumbar enlargement of L4 –L6 was obtained, immediately frozen and stored at –70 °C. Collected tissue samples were homogenized in lysis buffer containing a mixture of protease inhibitors. Protein concentrations were determined, and aliquots (10 μg) were loaded and run on 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel, and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked with 5 % non-fat milk in Tris-buffered saline (pH 7.5) containing 0.1 % Tween 20 for 1 h, and incubated overnight at 4 °C with rabbit anti-P2X7R (1:1000; Millipore).
After washing, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:1000; Cell Signaling, USA) for 2 h at 4 °C, developed in enhanced chemiluminescent solution (Millipore) for approximately 1 min, and exposed onto film. Densities of specific P2X7R bands were using a computer-assisted imaging analysis system and were normalized against corresponding β-actin (1:1000; Cell Signaling) levels as a sample loading control.