Anti nf κb sc 8008

Immunofluorescence and immunohistochemistry assays were performed as previously described [2 (link), 10 (link)]. The following primary antibodies were used: anti-Cx43 (Sigma-Aldrich, C6129), anti-collagen II (Invitrogen, Thermo Fisher Scientific, MA5-12789), anti-Ki-67 (BD, 550609), anti-Twist-1 (sc-81417) and anti-NF-κB (sc-8008) from Santa Cruz Biotechnology. Goat anti-rabbit FITC-conjugated (F-2765, Invitrogen, Thermo Fisher Scientific) and goat anti-mouse Alexa 594-conjugated (A-11032, Invitrogen, Thermo Fisher Scientific) secondary antibodies were used.

To block endogenous peroxidase activity, 5 μm-thick deparaffinized sections were incubated with 1% H₂O₂ for 30 min at room temperature. To observe the morphological characteristics and detect protein expression in the quadriceps muscle, the quadriceps muscle tissues were reacted overnight at 4 °C with anti-TWEAK (ab37170; 1:400; Abcam, Cambridge, UK), anti-NF-κB (sc8008; 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PGC-1α (bs-1832R, 1:300, BIOSS, Beijing, China) and anti-MuRF1 (bs-2539R; 1:300; BIOSS, Beijing, China) antibodies. Samples were then washed with PBS and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (PV-8000; 1:200; ZSGB-BIO, Beijing, China) for 1 h at room temperature. Following the removal of non-reacted secondary antibodies by washing with PBS, samples were incubated with 3,39-diaminobenzidine (DAB; Sigma-Aldrich; Merck KGaA) in a DAB-4HCl-H₂O₂ solution to visualize immunolabeling. Some sections were also counterstained with hematoxylin and eosin and mounted with a coverslip with neutral resins. Immunohistochemical analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). A total of 4–5 images that were positive for protein expression were randomly selected and their integral luminosity values and average optical density were assessed using a light microscope.

Anti-PC2 (sc-47734; for immunoblotting), anti-NFκB (sc-8008), and anti-IκBα (sc-1643) antibodies were purchased from Santa Cruz Biotechnology. Anti-PC2 antibody for immunofluorescent imaging was a kind gift from Dr. Stefan Somlo (Yale). Mouse anti-beta-Actin (#3700), rabbit anti-beta-Actin (#4970), anti-CHOP (#2895), anti-cleaved caspase-3 (#9664), and anti-GAPDH (#2118) antibodies were purchased from Cell Signaling Technology. Anti-4-Hydroxynonenal (ab48506), anti-eIF2α (ab50733), anti-phospho-eIF2α (ab32157) antibodies, and kainic acid (ab120100) were purchased from Abcam. Anti-VDAC (PA1-954A) antibody was purchased from Thermo Fisher Scientific. Hydrogen peroxide (H325-100) and BAPTA-AM (B1205) were purchased from Thermo Fisher Scientific. Tunicamycin (T7765), and Dimethyl sulfoxide (DMSO; W387520) were purchased from Sigma-Aldrich.