Anti mouse secondary antibodies

Genistein (99% purity; #446-72-0), purchased from Pharmaceutical Research Institute in Warsaw (Poland), was diluted in DMSO at stock concentrations of 30, 60, and 100 mM and stored at − 20 °C. Plasmid pEGFP (encoding EGFP) was from Addgene (#6077-1), as were plasmids pEGFP-Q74 (encoding EGFP protein fused to a fragment of huntingtin corresponding to the exon 1 with 74 CAG repeats; c.54GCA[74]; Addgene; #40262) and pEGFP-Q23 (encoding EGFP protein fused to a fragment of huntingtin corresponding to the exon 1 with 23 CAG repeats; c.54GCA[23]; Addgene; #40263) (these plasmids were gifts from Dr. David Rubinsztein to Addgene). Antibodies against GFP (used at dilution 1:4000) were from Santa Cruz Biotechnology (#sc-9996), and those against LC3 (used at dilution 1:2000) from MBL International (#PM036). The anti-mouse secondary antibodies (used at dilution 1:4000), as well as anti-β-actin antibodies (used at dilution 1:25,000) conjugated with HRP (#A3854), were from Sigma-Aldrich. Lysotracker Red was purchased from Life Technologies (#L-7528). Muse^® Annexin V and Dead Cell Assay Kit were purchased from Merck (#MCH100105).

Firstly, the protein was extracted by 1×EBC lysate and 2xSDS, and the protein expression level was analyzed by Western Blot: 10 μl denatured protein samples were added to each well, electrophoresis with 10% SDS-PAGE, and protein separation completely was performed with 1 x running buffer at 100 V (28 (link)–30 (link)). The protein was transferred to the PVDF membrane at 100V for about 90 min. Then sealed the PVDF membrane at room temperature with 5% milk for 2h, and incubated the primary antibody with 2% milk at 4°C overnight. Primary antibodies included CTSL antibody (1:10000, Abcam Catalog No. ab200738), β-actin antibody (1:5000 Cell Signaling Technology), and HSP70 antibody (1:2000 Sigma USA). On the second day, PVDF (polyvinylidene fluoride) membrane was washed three times with 1×TBST, and the secondary antibody was incubated at room temperature for 1-2h. The secondary antibodies included anti-rabbit secondary antibodies (1:2000) and anti-mouse secondary antibodies (1:2000) which were purchased from Sigma (USA). The PVDF membrane was then cleaned with TBST. An imaging scanner was used to detect the strength of each cell membrane strip after TBST washing three times. Each experiment was repeated three times.