Super signal west pico plus sensitivity substrate

HEK293 cell pellets or whole retinas were lysed in a lysis buffer containing 1% (w/v) SDS, 10 mM Tris, and 1 mM EDTA, pH 8.0. Following sonication, lysates were cleared by centrifugation at 18,000 g for 10 min and protein concentration was determined by the Pierce BCA assay (Thermo Fisher Scientific, Rockford, IL). Equivalent amounts of protein samples were separated on 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk (Bio-Rad, Hercules, CA) and incubated with primary antibody diluted in 3% milk overnight, followed by horseradish peroxidase-conjugated secondary goat anti-mouse or donkey anti-rabbit IgG for 1 h at room temperature. Immunoreactivity was detected by chemiluminescence using Super-Signal West Pico Plus Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL). Membranes were reprobed as necessary for the various markers. Primary antibodies used were as follows: anti-ELOVL4 at 1:1,000 (5 (link)), anti-MYC #2276 at 1:5,000 (Cell Signaling Technology, Waltham, MA), anti-HA#2367 at 1:2,000 (Cell Signaling Technology, Waltham, MA), anti-β-Tubulin #66240-1 at 1:10,000 (Proteintech, Rosemont, IL), and anti-GFP # ab290 at 1:3,000 (Abcam, Waltham, MA).

Cell pellets were harvested and resuspended in lysis buffer (600 mM NaCl, 1% Triton X-100 in PBS, 1 × protease inhibitor). The protein extracts were separated by electrophoresis in a 12% polyacrylamide gel before transferring to PVDF membrane. Membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature. Incubation with primary antibodies was performed at 4°C overnight (E2F1, ABclonal, catalog no. A19579; MYC, ABclonal, catalog no. A17332; TERT, ABclonal, catalog no. A2979; V5, Invitrogen, catalog no. R960-25; E-Cadherin, Cell Signaling Technology, catalog no. 14472; N-Cadherin, Cell Signaling Technology, catalog no. 14215; GAPDH, ABclonal, catalog no. AC002). Membranes were washed three times with TBST every 10 min before secondary incubation with antibodies fused to horseradish peroxidase (HRP) (goat anti-mouse IgG, ABclonal, catalog no. AS003; goat anti-rabbit IgG, Thermo Fisher Scientific, catalog no. 31460) for 1 h at room temperature. After three final washes, Chemiluminescence signal was developed with SuperSignal™ West Pico Plus Sensitivity Substrate (Thermo Fisher Scientific, catalog no. 34094).

Three of each cell-seeded mineralized collagen and mineralized collagen–amnion scaffold were lysed for protein at Days 0, 4, 7, 14 and 28. To extract protein, a solution of phosphate inhibitor cocktails (Sigma-Aldrich) and an RIPA lysis buffer were used [42 (link)]. To evaluate the protein concentration, a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) and a Pierce^TM Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Fisher Scientific) were used. Analysis of protein activity was quantified with a Western Blot assay, loading 5 µg of protein lysate in each lane and using a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) or a SuperSignal West Pico PLUS Sensitivity Substrate (Thermo Fisher Scientific) to visualize bands. Images were captured with an Image Quant LAS 4010 machine (GE Healthcare Life Sciences, Little Chalfont, UK). Primary and secondary antibodies are listed in Table 1, and β-actin was used as a loading control.