Omni ecl femto light chemiluminescence kit

For Western blot analysis, cells were lysed with 1% TritonX/PBS + 1 mM PMSF (Beyotime, ST506) for 10 min at 4 °C, then clarified through centrifugation of 12,000 rpm for 5 min at 4 °C. The clarified cell lysate was mixed with the 1/5 volume of 5× SDS loading buffer and incubated at 98 °C for 10 min. After gel electrophoresis and membrane transfer, the PVDF-membrane blots were blocked with 5% skimmed milk in PBST for 2 h at room temperature and then incubated 1 μg/mL anti-Flag mAb (Sigma, F1804), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AntGene, ANT325) PAb or anti-β-tubulin (Immmuno Way, YM3030) mAb diluted in PBST containing 1% milk overnight at 4 °C. After three times washing with PBST, the blots were incubated with Horseradish peroxidase (HRP)-conjugated secondary antibody AffiniPure Goat Anti-Mouse or Rabbit IgG (H + L) (Jackson Immuno Research, 115-035-003 or 111-035-003) in 1% skim milk in PBST and incubated for 1 h at room temperature. The blots were then washed three times by PBST and then visualized using an Omni-ECL Femto Light Chemiluminescence Kit (EpiZyme, SQ201) by a ChemiDoc MP Imaging System (Bio-Rad).

Mice were subjected to deep anesthesia by urethane, followed by transcardial perfusion with cold PBS. Ipsilateral L4 ~ L5 DRGs and SDHs were carefully dissected, homogenized, and sonicated in a lysis buffer containing a protease inhibitor cocktail (Cat# P1045, Beyotime Biotechnology). For protein analysis, 15 μg total proteins were loaded into each well, separated using SDS-PAGE, and subsequently transferred to PVDF membranes. The membranes were blocked with a solution of 5% bovine serum albumin in TBST (Tris-buffered saline, 0.1% Tween 20) at RT for 1 h. Next, they were incubated overnight at 4 °C with primary antibodies including Rb anti-p-ERK (#4370, Cell Signaling Technology, 1:1000), Rb anti-TRPA1 (SAB2105082, Sigma, 1:1000), Rb anti-TRPV1 (GTX54762, Genetex, 1:200, and Ms anti-β-actin (ab170325, Abcam, 1:1000). Subsequent steps involved incubating the blots with horseradish peroxidase-conjugated IgG at RT for 1 h. Protein bands were detected by Omni-ECL™Femto Light Chemiluminescence Kit (SQ201, Epizyme) and then captured by Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology, China). The integrated optical density of each immunoreactive band was measured Software ImageJ 1.51j8, and then normalized to β-actin. The protein X/β-actin ratio in the saline group was established as the baseline for comparison.

The samples were added 100 μL RIPA (Solarbio) cell lysate containing 1 mM PMSF (Solarbio) and incubated on ice for 30 min. Cells were scraped evenly, and the lysate was collected into an EP tube. After centrifugation at 4000 ×g for 10 min, the supernatant was obtained, and the protein concentration was determined by the BCA method (Beyotime). Then SDS-PAGE gel electrophoresis was performed after adding loading buffer into the supernatants. The following proteins were transferred to a PVDF membrane (.22 μm; Millipore, Billerica, MA, USA). The primary antibody (anti-Bax, anti-Cleaved Caspase-3, anti-β-actin, anti-GAPDH, Abcam; anti-MAP4K4, Immunoway; anti-Bcl-2, anti-t-ERK, anti-p-ERK, anti-t-JNK, anti-p-JNK, anti-t-p38, anti-p-p38, CST; anti-pORF5 [22 (link),23 (link)], MAb clone 2H4) was incubated overnight after the PVDF membrane was blocked with 5% nonfat milk. Then the secondary antibody (HRP-conjugated goat anti-rabbit IgG, Abcam) was incubated after washing the membrane. Finally, ECL reagent (Omni-ECL™Femto Light Chemiluminescence Kit, Epizyme) was mixed and added to the PVDF membrane to avoid light. The results were visualized using an enhanced chemiluminescence western blot system G: BoxChemi X×X9 (Syngene, Cambridge, UK). Quantity One (BioRad, USA) analyzed the densities of protein bands.