Clarity ecl western blot substrate kit

Tau, GFAP, Iba1, and LRP1 protein levels in the SDS-soluble fraction of the hippocampus were semi-quantified by immunoblotting analysis. After blocking, membranes were incubated at 4 °C overnight with the corresponding primary antibody (HT7, 1:100, mouse monoclonal anti-human tau mAb; AT8, 1:50, mouse monoclonal anti-human phospho-tau (Ser202, Thr205); anti-GFAP, 1:2500; Iba1, 1:500; mouse monoclonal anti-LRP1 1:1000; abcam, ref. ab28320; and rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase, GAPDH, 1:20000, abcam, ref. ab9483). Membranes were incubated at RT for 1 h with the corresponding secondary antibody (goat peroxidase conjugated anti-rabbit, 1:2000, Bio-Rad, ref. 1706515; and goat peroxidase conjugated anti-mouse, 1:2000, Sigma-Aldrich Química SL, ref. A3673. Finally, Western blotting (WB) results were visualized using a lumiol/peroxidase solution 1:1 (Clarity ECL Western Blot Substrate kit, Bio-Rad, ref. 170-5060). ImageJ was used for densitometric analyses, and bands were normalized to GAPDH loading controls for each sample.

293T cells transfected as described above were lysed in the whole cell lysate buffer supplemented with protease inhibitors as described above. Total protein concentration was determined using BCA protein assay (Bio-Rad). Lysates were stored at -80 ℃ until use. Protein A/G agarose beads (Sana Cruz Biotechnology) were blocked with 5% Bovine Serum Albumin (BSA). Blocked beads were incubated with anti-Flag antibody then combined with protein lysate and incubated for 2 h at 4 °C. Beads were washed with 1x TNN buffer (10 mM Tris-HCl 7.5, 0.1% NP-40 and 100 mM NaCl) and then heated for 5 min at 95 °C. Eluted proteins were loaded on 10% Bis-Tris gel and resolved in 1x MOPS running buffer supplemented with SDS and anti-oxidant (Invitrogen). Proteins in the gel were transferred to PVDF membrane. The membrane was blocked in 5% non-fat milk, washed in PBS buffer containing 0.1% Tween-20 (PBST) and incubated with primary antibodies including Anti-CDK9 (1:4000) (Rabbit, Lot No: I0414), Anti-Cyclin T1 (1:4000) (Rabbit, Lot No:D1709) or anti-Flag 1:2000 for 2 h at room temperature. The membrane was washed with PBST and incubated with HRP-conjugated anti-mouse or anti-rabbit and probed. Chemiluminescence was detected with chemiluminescent substrate (Clarity ECL Western Blot Substrate Kits, Bio-Rad) using ChemiDoc XRS Imaging station (Bio-Rad).

Proteins were extracted using an ice-cold RIPA lysis buffer (P00138, Beyotime, Nanjing, China) with proteinase inhibitor (ST506, Beyotime, Nanjing, China). Equal amounts of proteins were measured using an BCA protein assay kit (A045-3, Jiancheng, Nanjing, China). Proteins were separated by electrophoresis and then electronically transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, USA) using a BioRad system (BioRad, Hercules, USA). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and subsequently incubated overnight at 4°C with corresponding primary antibodies, rabbit anti-BCL2 (1:200), mouse anti-GRP78 (1:200), rabbit anti-LC3β (1:100, sc-28266, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-CYP11A1 (1:200, CSB-PA006389LA01HU, CusAb, Wuhan, China), mouse anti-BAX (1:100, ab5714) and mouse anti-β-actin (1:1000, ab8226, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (sc-2004 or sc-2005, Santa Cruz Biotechnology, Dallas, USA) were then used to detect proteins using Clarity ECL Western Blot Substrate kits (BioRad, Hercules, USA) and exposed using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). Protein quantification was performed using densitometry analyses on Quantity One Software.