Protein Expression Analysis by Western Blot

Protein levels were detected by western blotting referring to the protocols in a previous report [20 (link)]. In brief, HaCaT cells were lysed in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China), and extracted protein was quantified by bicinchoninic acid assay kit (Thermo Fisher Scientific). Protein samples (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transfer of polyvinylidene fluoride membranes (Thermo Fisher Scientific). After incubating in 5% fat-free milk, the membranes were incubated with primary antibodies for IGF2BP2 (ab129071, 1:3000 dilution, Abcam, Cambridge, UK), HPSE (ab254254, 1:3000 dilution, Abcam), or β-actin (ab8227, 1:3000 dilution, Abcam) overnight, and then incubated with horseradish peroxidase-conjugated IgG (ab205718, 1:8000 dilution, Abcam) for 2 h, followed by exposure to enhanced chemiluminescence kit (Beyotime). The visualized blots were analyzed via Image J software (NIH, Bethesda, MD, USA), with β-actin as a reference.