Culture cell total protein extraction reagent

The protein lysate was obtained from culture cells by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) with protease inhibitor and phosphatase inhibitor cocktail. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). Primary antibodies included rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Occludin (#27260–1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN) and rabbit anti-phospho-NF-κB p65 (#3033 T; CST, Danvers, MA USA). Goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN) was used as secondary antibody.

PC12 cells were seeded in 6-well plates at 4.0×10⁵ cells per well, treated with X5 for 18 h, and sampled for total proteins on ice using the culture cell total protein extraction reagent (Boster Biological Technology Co. Ltd., China). Total protein was then separated by SDS-PAGE electrophoresis and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then incubated with primary antibodies (Anti-SIRT1, Proteintech, 13161-1-AP, China, 1:1000; anti-beclin-1, Proteintech, 11306-1-AP, China, 1:1000; anti-LC3-β, Proteintech, 18725-1-AP, China, 1:1000; anti-GAPDH, Proteintech, 10494-1-AP, China, 1:1000) at 4°C for overnight. The following day, the membrane was incubated with secondary antibodies (Anti-Rabbit igg (H + L), Proteintech, SA00001-2, China, 1:2000) at room temperature for 90 min, and the immunofluorescent bands were imaged using the ChemiDoc XRS+ system (Bio-Rad, USA). Finally, the images were quantitatively analyzed using Image J (NIH, USA).

The protein lysate was obtained from tissue by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) and mammalian tissue protein extraction reagent (BOSTER, Wuhan, CHN), with protease inhibitor and phosphatase inhibitor cocktail, respectively. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). The primary antibodies included the following: rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-occludin (#27260-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN), rabbit anti-phospho-NF-κB p65 (#3033T; CST, Danvers, MA USA), rabbit anti-p38 (#ab27986; Abcam, Cambridge, MA, USA), and rabbit anti-phospho-p38(#ab47363; Abcam, Cambridge, MA, USA). The secondary antibody used was goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN).