High efficiency ripa tissue cell lysis buffer

Tris, glycine, 30% acrylamide, sodium dodecyl sulfate (SDS), Tween-20, PMSF, and high-efficiency RIPA tissue/cell lysis buffer were purchased from Solarbio Science & Technology Company (Beijing, China). Anti-cathepsin Z (CTSZ; #ab182575), anti-C3 (#ab200999), anti-CD11b (#ab133357), anti-CD16 (#ab211151), and anti-NeuN (#ab104224) antibodies were purchased from Shanghai Abcam Company. Anti-GBP2 antibody (#11,854–1-AP) was purchased from Proteintech. Anti-GFAP (#MA5-12,023), anti-β-actin (#MA5-15,739), anti-β-tubulin (#MA 5–11,732), Alexa Fluor 555-conjugated goat anti-mouse IgG (#a21424), and Alexa Fluor 488-conjugated goat anti-rabbit IgG (#a11034) antibodies were purchased from Invitrogen. Enhanced chemiluminescence (ECL) Western blot detection reagent (#32,106) was purchased from Thermo Fisher Scientific, and 2,3,5-triphenyltetrazolium chloride (TTC, #T8877-25G) was purchased from Sigma. A Fluoro-Jade C Kit (FJC, TR-100-FJT) was purchased from Biosensis.

Cell lysates were prepared using high-efficiency RIPA tissue/cell lysis buffer (Solarbio, Beijing, China), and then the protein content was quantified using the BCA protein assay kit (Solarbio, Beijing, China). SDS-PAGE gel electrophoresis was used to separate the same amount of protein samples and then transferred to PVDF membrane on ice (Solarbio, Beijing, China). After washing three times with TBST (10 min each) (Solarbio, Beijing, China) and then using a commercial enhanced chemiluminescence (ECL) kit (Solarbio, Beijing, China) for visualization. All antibodies are from Sant Cruz company products, the antibody source is rabbit antigen, and the primary antibody ratio is 1:200; the secondary antibody is mouse-to-rabbit, and the ratio is 1:1000.

MDA-MB-231 and MDA-MB-468 cells were lysed and proteins were extracted using high-efficiency RIPA tissue/cell lysis buffer (Solarbio, Beijing, China). Antibodies used were as follows: KCNQ4 antibody (1:1000, Abways, Shanghai, China), PI3K antibody (1:1000, Abways, Shanghai, China), AKT1/2/3 antibody (1:1000, Abways, Shanghai, China), p-AKT antibody (1:1000, Abways, Shanghai, China) and GAPDH antibody (1:5000, CUSABIO, Wuhan, China). Exposure was performed using a chemiluminescence gel imaging system.