Luminata crescendo western hrp substrate

The collected liver tissues from different groups were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer to extract total proteins. Total protein concentration was determined using the BCA Protein Assay Kit. Then the proteins were loaded and separated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and then transferred to polyvinylidene fluoride membranes (Millipore). After soaking in blocking liquid for 2 h, polyvinylidene fluoride membranes were transferred to primary antibodies solution and incubated at 4°C overnight to react with Bcl-2 (1:500), cleaved caspase-3 (1:500), Bax (1:1000), and GAPDH (1:2000), respectively. Then the membranes were washed with Tris-buffered saline with Tween-20 (TBST) three times, transferred to secondary antibodies (1:4000) solution for 2 h at room temperature. They were washed with TBST again, and then Luminata Crescendo Western HRP Substrate (Bio Rad, Berkeley, CA, USA) was added. The membranes were exposed in a gel imaging analysis system (Alpha Innotech FluorChem FC2, USA). Images were analyzed using ImageJ software to determine gray scale. Samples were assessed in triplicate using different batches samples.

Triplicates of Tpm3.1-APEX2 +/− and −/− PMEFs were grown on 6 cm dishes until full confluency was reached. Cell lysates were harvested in 4°C RIPA buffer with protease inhibitor (cOmplete, EDTAfree Protease Inhibitor Cocktail, Merck) and homogenized by sonication for 30 s. Protein concentration was measured with Precision Red Assay (Cytoskeleton). Laemmli sample buffer (Biorad) was added 1:4 (v/v) and lysates were boiled at 95°C for 10 min. Samples were run at 100 V for 90 min on 10% polyacrylamide SDS-PAGE gels in running buffer. Gels were semi-dry-transferred in transfer buffer to PVDF membranes preactivated with 100% methanol. Membranes were blocked in 5% skim milk in TBS for 1 hr and probed with mouse γ/9d 2G10.2 (1:1000, MERCK MABT1335), rabbit anti-mouse IgG (1:3000, Abcam ab97046), rabbit α-tubulin (1:3000) (Abcam ab52866) and goat anti-rabbit IgG (1:5000) (Biorad 170-6515) antibodies sequentially for 1 hr. Luminata Crescendo Western HRP substrate (Merk) was used for imaging on a Chemicoc MP imaging system (Biorad). Band densitometry was quantified (ImageJ) and normalized to α-tubulin control.