Immobilon ecl hrp substrate

Cells were seeded at a density of 1 × 10⁶ cells/plate on 60 mm cell culture dish overnight. Cells were incubated with M5 or PG102 at designated concentrations for 30 min and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents in the cell lysates were determined by bicinchoninic acid (BCA) assay kit and after reconstituting in sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane (Merck) and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 h to block nonspecific binding. The membrane was then incubated with antibodies specific for phospho-STAT3 (#9134, #9145), STAT1 (#9167), p65 (#3033), and STAT3 (#4904), STAT1 (#9172), p65 (#8242), IκB-α (#9242) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (A5441, Sigma-Aldrich) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1:100,000; Sigma-Aldrich) at room temperature for 1 h. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure on autoradiography film.

HaCaT cells were seeded at a density of 4.5 × 10⁵ cells/well onto 6-well plates overnight. Cells were then treated with PG102 at designated concentrations for 48 hours, and total cell lysates were extracted with CytoBuster™ (Merck, Darmstadt, Germany) mixed with PhosSTOP™ and cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Total protein contents were measured by the BCA assay kit (Invitrogen), and after reconstitution in a sample buffer, 20 micrograms of protein samples was subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (Invitrogen). Proteins were transferred onto a PVDF membrane, and the membrane was incubated in 5% skim milk in 0.1% TBST at room temperature for 1 hour to block nonspecific binding. The membrane was then incubated with antibodies specific to IL-37, phopho-Smad3 (1 : 1000; Abcam), Smad3, phospho-ERK, ERK (1 : 1000; Cell Signaling Technology, Danvers, MA), and β-actin (1 : 5000; Sigma-Aldrich) overnight at 4°C followed by incubation with a horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit IgG (1 : 100,000; Sigma-Aldrich) at room temperature for 1 hour. The blot was developed by Immobilon ECL HRP substrate (Merck) and visualized by exposure to autoradiography film.

Total protein from TNBC cells was extracted by RIPA lysis buffer (Beyotime, Nantong, China) and quantified with a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amount of protein (30 μg) was separated on 10% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), and then were probed with primary antibodies against Ki-67 (1:1000; ab243878, Abcam, Cambridge, MA, USA), CDCA3 (ab166902; 1:1000, Abcam), and GAPDH (1:5000, ab70699, Abcam) at 4 °C overnight. A HRP-conjugated secondary antibody was further used to incubate with the membranes at room temperature for 1 h. Finally, the protein bands were visualized by Immobilon ECL HRP substrate (Millipore, Billerica, MA, USA).