Ab92333

Total protein content was assessed using a bicinchoninic acid (BCA) kit (Bosterbio, Pleasanton, USA). An equal amount of protein was used in SDS-PAGE. Proteins were transferred onto the polyvinylidene fluoride (PVDF) membranes (Biocompare, San Francisco, USA) after electrophoresis, followed by the blocking with 5% skim milk powder in Tween and Tris-buffered saline (BioLegend, San Diego, USA) for 50 min. These membranes were incubated with primary antibodies including anti-Capn4 (ab92333; 1:2000; Abcam, Cambridge, USA), anti-NF-κB (#8242; 1:1000; Cell Signaling, Beijing, China), anti-phospho-NF-κB (ab183559; 1:1000; Abcam), anti-IKK-β (ab124957; 1:1000; Abcam), anti-p-IKK-β (ab194519; 1:1000; Abcam), and β-actin (ab8227; 1:2000; Abcam) at 4°C overnight followed by the incubation with Goat anti-Rabbit horseradish peroxidase (HRP) (ab7090; 1:2000; Abcam) at 37°C for 1 h. β-actin was the internal control. The chemiluminescence reagent (Thermo Fisher Scientific) was used to visualize the bands and detected under a Las-3000 imaging system (Biocompare).

Protein concentration was determined using a BCA kit (Thermo Scientific). The SPEs sample proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Proteins were then subjected to immunoblot. All primary and secondary antibodies were diluted in PBS. Primary antibody incubation was performed at 4°C overnight, and secondary antibody incubation was performed at room temperature for 2 h. The immunoreactive blots were visualized using an enhanced chemiluminescence reagent. Antibodies against CAPNS1 (rabbit monoclonal, 1/2000, ab92333), TLN1 (rabbit polyclonal, 1/400, ab71333) was purchased from Abcam (Cambridge, Cambs, UK). Antibodies against SRC (rabbit polyclonal, 1/1000, CST2108), ALDOA (rabbit polyclonal, 1/1000, 3188S) and ITGB2 (rabbit polyclonal, 1/1000, CST4702) were purchased from Cell Signaling Technology. Cd63 (rabbit polyclonal, 1/200, sc-15363), Annexin1 (mouse monoclonal, 1/200, sc-7964), HSP27 (mouse monoclonal, 1/5000, sc-13132) were purchased from Santa Cruz Biotechnology. We used Coomassie Brilliant Blue staining as a loading control. Densitometric analysis was performed on the Western blot images using ImageJ software (nih.gov).