Quantifying p62 Protein Expression by Western Blot

As previously described [14 (link)], cells from each group were collected and placed in a 1.5 mL EP tube, followed by addition of appropriate amount of lysate for completely lysis and ultrasonic testing in the ice bath sing ultrasonic processor; after centrifugation at 2000 rpm for 15 min at 4 °C, the supernatant was extracted and stored at − 20 °C for further use. The oncentration of proteins extracted above was quantified sing BCA Protein Quantification Kit; 5 × loading buffer as added to the sample, followed by boiling at 100 °C or 5 min to denature the proteins. After processing by DS-PAGE, the proteins were transferred to the NC embrane using protein transfer device and then locked by 5% skimmed milk powder for 1 h. After addition of p62 antibodies (Abcam UK) and incubation vernight at 4 °C, the proteins were washed by BST three times before 1 h of fluorescent secondary ntibody incubation and another three times of TBST ashing; Western Blot imaging software Odyssey nalyzer was used to detect and produce images.