Protease and phosphate inhibitors

The tissue samples of testis were homogenized in an ice-cold RIPA buffer containing protease and phosphate inhibitors (Roche Applied Science, Penzberg, Germany). The concentration of protein in the supernatant was measured using the bicichonic acid assay after centrifugation at 13,000 g for 15 mint, protein samples 10μl were loaded onto SDS-PAGE gels at 4-12%. Electrophoresis was performed at 120 V for 2 hr at 4° C (Bio-Red Mini-Protein), followed by immunoblotting with primary antibodies dilution (1:1000) and Actin (1:10,000) (Table 2). Targeted proteins intensities CD63, LC3, P62 and LAMP2 were normalized against actin. Western blotting quantitative measurements were performed in three independent experiments.

Small intestinal tissue samples were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer that contained protease and phosphate inhibitors (Roche Applied Science, Penzberg, Germany). After centrifugation at 13,000× g for 15 min, the protein concentration in the supernatant was quantified with the bicinchoninic acid protein assay (Thermo Scientified). Protein samples (45 µg) were loaded on 4–12% SDS–PAGE gels. Electrophoresis was run at 120 V for 2 h at 4 °C (Bio-Red Mini-Protean), and immunoblotting was performed with primary antibodies (diluted 1:1000) and β-actin (1:10,000; Bioworld, Nanjing, China). The intensities of target proteins LC3, p62, PINK1, and AMPK were normalized against β-actin. Three independent experiments were performed for western blotting quantification analysis.

Cells were washed in PBS and lysed in 1X RIPA (Millipore) with protease and phosphate inhibitors (Roche). Lysates were cleared by centrifugation, and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Fifteen to twenty micrograms of protein was boiled in 2X sample buffer and then resolved on 4–12% gels (Biorad). Protein was transferred to nitrocellulose (GE Healthcare) and blocked in TBS-T (0.1% tween) with 5% milk for at least 1 hour. Primary antibodies were added overnight in blocking buffer. The following days blots were washed for 1 hour in TBS-T, and then secondary antibodies were added in blocking buffer for 1 hour. Blotted protein was visualized using Thermo ECL reagents. Western blots performed on serum starved MEFs were performed by pooling several 10 Cm dishes of approximately 0.7*10⁶ cells (40–60% confluent) control and Kif7 mutant MEFs due to low protein concentrations. Cell fractionation was performed as described by [53 (link)]. Co-immunoprecipitation of GFP-fused KIF7 was performed using the GFP trap kit (Chromotek) according to the manufacturer instructions. Quantification of bands from immunoblots was performed using Image J software.