Ab 154894

Proteins were obtained using radioimmunoprecipitation assay (RIPA) buffer to dissolve the cells. The quantification was tested using bicinchoninic acid Protein Assay Kit (Boster Biological Company, Ltd., Wuhan, China). All protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The target strip was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA) via electrophoresis. The PVDF membrane was blocked in 5% nonfat milk. The protein bands were incubated with specific primary antibodies as follows: HNRNPH1 (1:2,000) (ab 154894, Abcam, CA, USA), PTPN6 (1:1,000) (ab 32559, Abcam, CA, USA), AKT (1:1,000) (ab 38449, Abcam, CA, USA), p-AKT (1:1,000) (ab 8805, Abcam, CA, USA), and β-ACTIN (1:8,000; Abways Technology, New York, NY, USA; AB0035). Moreover, the PVDF membrane was incubated in a goat-anti-rabbit secondary antibody (1:10,000, Boster Biological Company, Ltd., Wuhan, China) overnight. Images of protein quantification were captured by BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA).

Mice brains total protein extracts were separated by SDS-PAGE and then transferred onto nitrocellulose membranes (Bio-rad, Hercules, California, US). Membranes were blocked with 5% non-fat milk and then incubated with the following antibodies: rabbit polyclonal anti-hnRNPH (Abcam, ab154894), rabbit polyclonal anti-SERPIN B6 (Abcam, ab233229; Proteintech, 14962-1-AP), rabbit polyclonal anti-IRGM (Abcam, ab118569), rabbit polyclonal anti-UQCRQ (Proteintech, 14975-1-AP), rabbit monoclonal anti-HOMER1 (Abcam, ab184955), rabbit monoclonal anti-HTT (Cell Signalling Technology, mAb#5656), rabbit polyclonal anti-OSBPL2 (Proteintech, 14751-1-AP), mouse monoclonal anti-β-Actin (Origene, TA811000) and mouse monoclonal anti-vinculin (Sigma-Aldrich, V9131). Membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 for mouse host antibodies and 1:10000 for rabbit host antibodies) for 45 minutes at room temperature and the signals were detected by enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Inc., Waltham, MA).
For densitometric analyses, the software Quantity One 4.6.8 (Bio-Rad, Hercules, California, US) has been employed; all protein band intensities were normalized with the corresponding β–actin signal, except HTT, whose intensities were normalized on vinculin signal.