Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page gel

After treatment with different concentrations (0, 10, 25, 50, and 100 μg/mL) of PM_2.5 for 24 h, the HaCaT cells were rinsed twice in phosphate buffered saline (PBS). Then, the protein extracts were obtained by cell lysis buffer (Beyotime, Haimen, China) and spun at 14,000× g for 10 min at 4 °C. Total proteins for each sample were loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime). After electrophoresis, proteins were transferred onto a nitrocellulose membrane. Blots were rinsed twice in Tris-buffered saline–Tween (TBST). After being blocked for 2 h at room temperature in TBST plus 5% skim milk powder, the nitrocellulose membrane was incubated with different dilutions of primary antibodies—FLG, LOR, IVL, Repetin (RPTN), or β-actin (Abcam, Cambridge, UK)—over 12 h at 4 °C. Then, the membrane was rinsed three times in TBST (10 min each at room temperature) and incubated for 2 h at room temperature with a secondary antibody (Beyotime). Blots were finally rinsed clearly and detected by Immobilon Western (Millipore, Boston, MA, USA). The protein bands were scanned with a LAS3000 imaging system (Fujifilm, Tokyo, Japan), and band density was calculated by Quantity One software (Bio-Rad, Hercules, CA, USA). β-actin was used as a control.

Total cellular proteins were extracted from AFCs at the end of the mechanical loading period using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) according to the manufacturer's protocol. These proteins were quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Extracted proteins were denatured and separated in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime, Shanghai, China), and then transferred by electrophoresis onto polyvinylidene fluroride (PVDF) membranes (Beyotime, Shanghai, China). Membranes were blocked in blocking buffer for 60 min and incubated with properly diluted primary antibodies at 4 °C overnight (All antibodies used for Western blot analysis were diluted as 1:1000). The membranes were washed and then incubated with respective horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Proteins were detected by autoradiography (Bio-Rad, Hercules, CA, USA) and the grayscale value was quantified using ImageJ software (NIH, Bethesda, MD, USA). The primary antibodies used in this study were listed in Table 2.

Total protein was collected with the RIPA buffer (Beyotime). And the concentration of these proteins was measured with the BCA (Beyotime) method. Next, these proteins were separated with the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime). After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). Then these membranes were blocked with the 5% skim milk powder and incubated with the primary antibodies at 4 °C overnight. The primary antibodies used in this research were BNIP3 (ab109362, ABCAm), Bcl-2 (ab32124, ABCAm), Bax (ab32503, ABCAm), Cleaved caspase3 (ab49822, ABCAm), Cleaved caspase9 (ab2324, ABCAm), Little Computer 3 (LC3) I/II (ab62721, ABCAm), Autophagy-related protein 7 (ATG7) (ab133528, ABCAm), Beclin1 (#3495S, Cell Signaling Technology, USA), P62 (#5114S, Cell Signaling Technology), and GAPDH (ab8245, ABCAm). On the second day, these membranes were washed with the phosphate-buffered solution (PBST) for three times and then incubated with the second antibody for 2 h at room temperature. At last, these membranes were washed with the PBST again and the immunoreactive signal was detected with the LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan).