Immun star hrp peroxide luminol enhancer

Cells were lysed in RIPA buffer (1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). Complete protease inhibitor cocktail (Roche, USA) was added to lysis buffer before use. Protein concentration was determined by Bio‐Rad DC protein assay (Bio‐Rad, USA). Protein samples were subjected to SDS‐PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% nonfat milk in PBST overnight and incubated with primary antibody and subsequently with appropriate horse radish peroxidase‐conjugated secondary antibody. Signals were detected using the Immun‐Star HRP peroxide Luminol/Enhancer (Bio–Rad, Cat. #1705040) and recorded on a ChemiDoc Touch Imaging System (Bio‐Rad, USA). Anti‐IL‐15Rα antibody was purchased from Santa Cruz (Dallas, USA, Cat. #sc‐374023). Anti‐Stat3 (Cat. #A1192), anti‐Jak3 (Cat. #A0748), antiphospho‐Jak3‐Y980/981 (Cat. #AP0532) and antiphospho‐Stat3‐Y705 (Cat. #AP0705) were purchased from ABclonal (Woburn, MA, USA).

One gram liver tissues from rats were homogenized using Tris-Triton cell lysis buffer (TTL buffer, 1% Triton X-100, 50 mM Tris-HCl pH 7.4, 5% glycerol, 100 mM NaCl) supplemented with protease and phosphatase inhibitor cocktails (Abcam). After adjusted to the same concentration, samples were boiled in SDS-PAGE sampling buffer, analyzed by 10% SDS-PAGE, and transferred onto the PVDF membrane. Immunoblots were probed with antibodies against Akt, phosphor-Akt (Ser473), Erk1/2 or phosphor-Erk1/2 (Thr408) (Cell Signaling Technology) and reprobed with anti-β-actin antibody (Sigma) to serve as a loading control. Signals of targeted proteins were detected by the Immun-Star HRP peroxide Luminol/ Enhancer (BIO-RAD) and recorded on CL-XPosure Film (Thermo Scientific). The gray scale of the band was measured by Quantity One image analysis software (Bio-Rad) and the relative expression of the target protein was calculated with the net index (NI) of the β-actin. The relative expression of the target protein = target protein gray value / β-actin gray value. The level of phosphorylation was expressed as a percentage of p-Akt / Akt or p-ERK1/2 / ERK1/2 gray values, respectively.