Mouse anti bcl 2

Immunofluorescence assays were performed as described previously [52 (link)]. After adding the primary antibodies (mouse anti-Bcl-2(Abcam), mouse anti-Mcl-1(Abcam)), we added appropriate secondary antibodies (FITC-goat anti-mouse IgG Santa Cruz) to the samples. DAPI was used to stain nuclei. The samples were then evaluated under an inverted laser scanning confocal microscope (OLYMPUS-FV-1000, Japan).

The cells were lysed with a lysis buffer (Beyotime, Haimen, China), and centrifuged at 12,000 × g for 20 min. The total protein concentration was determined using the a BCA protein assay kit (Pierce). Samples were resolved in SDS-PAGE, transferred to 0.45 μm nitrocellulose transfer membranes and analyzed separately. After blocking with 5 % skim milk at room temperature for 60 min, the blots were survey with primary antibodies against mouse anti-Bcl-2 (1:500; Abcam), rabbit anti-LC3 (1:1000; Abcam), mouse anti-GAPDH (1:1000; Cell Signal), and rabbit anti-active caspase-3 (1:1000; Abcam), at 4 °C for overnight. We washed the membranes three times with TBST buffer (20 mmol/L Tris-buffered saline and 0.1 % Tween 20) for 1 h. And we used peroxidase conjugated anti-mouse-IgG/anti rabbit-IgG as secondary antibodies. We did Chemiluminescence with Amersham ECL plus Western blotting detection system (GE healthcare). After washing with the TBST buffer, the membranes were measured with the Odyssey Infrared Imaging System (LI-COR).

The cells were sonicated for 1 minute in the precooled RIPA lysate (Beyotime) and centrifuged at 12,000 g for 10 minutes at 4°C and the supernatant was stored at −80°C. The protein concentration in the sample was determined with BCA Protein Assay Kit (Beyotime). Electrophoretic separation was performed for the extracted protein with 10% SDS-polyacrylamide gel and the protein was transferred to PVDF (Millipore, Bedford, MA, USA). The membrane was sealed in TBST buffer containing 5% skim milk for 1 hour and then incubated overnight at 4°C with the following primary antibodies: anti-mouse-p-tlr4 (Santa Cruz, 1 : 1000), anti-mouse TLR4 (CST, 1 : 1000), anti-mouse-p-nf-κ B (Santa Cruz, 1 : 1000), anti-mouse-NF-κ B (Santa Cruz, 1 : 1000), anti-mouse-casepse3 (abcam,1 : 1000), anti-mouse-Bax (abcam,1 : 1000), anti-mouse-Bcl-2 (abcam,1 : 1000), and anti-mouse-GAPDH (Santa Cruz, 1 : 1000). Then the membrane was washed with TBST and incubated with horse radish peroxidase labeled goat anti-rabbit IgG (Beyotime, A0216, 1 : 5000) for 1 hour at room temperature. Enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) was used to detect protein bands, and Image Lab software (Bio-Rad) was used to quantify protein bands.