S0001

CSF1, CSF1R, and STAT1 protein levels in the induced sputum cells and BEAS‐2B cells were determined by western blot analysis according to the manufacturer's instructions. Abs to anti‐M‐CSF (1:1000; Abcam, ab233387), MCSF receptor (1:500; Affinity, AF0080), p‐CSF1R (1:500; Affinity, AF4395), STAT1 (1:500; Affinity, AF6300), p‐STAT1 (1:500; Affinity, AF3300), GAPDH (1:3000; Affinity, AF7021), Tubulin β (1:1000; Affinity, AF7011) and goat antirabbit IgG (H + L) HRP (1:4000; Affinity, S0001) were used in this study.

Detailed information about the Western blot analysis was previously described (Liang et al., 2022a (link)). The antibodies used are as follows: β-actin antibody (AF7018, Affinity), KIF20A antibody (AF7664, Affinity), Goat Anti-Rabbit IgG (H + L) HRP (S0001, Affinity) and Goat Anti-Mouse IgG (H + L) HRP (S0002, Affinity).

Total proteins were extracted from colon tissue or kidney tissue samples when treated with radioimmunoprecipitation assay (RIPA) lysis (Sangon Biotech, Shanghai, China). The concentrations of total proteins were measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred to an NC membrane. The NC membrane was blocked with 5% non-fat milk and then incubated with primary antibodies against ZO-1 (AF5145, 1:500, Affinity), occludin (CY5997, Abways), claudin-1 (AF0127, 1:1,000, Affinity), Col-I (AF0127, 1:2,000, Affinity), Col-III (AF0136, 1:1,000, Affinity), FN (CY5621, 1:1,000, Abways), and LN (CY6617, 1:1,000, Abways) overnight at 4°C, respectively. Then, the membrane was washed with TBST and incubated with secondary goat anti-rabbit (S0001, 1:5,000, Affinity) and goat anti-rat antibodies (S0009, 1:5,000, Affinity) at 37°C for 2 h (Cell Signaling Technology, CA, USA). Finally, the proteins were monitored with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, NJ, USA) and then quantitatively analyzed by ImageJ software (version 1.4.0., National Institutes of Health). GAPDH protein was used for the internal control.

Total cellular protein was extracted with the protein extraction kit (KeyGen Biotech, Nanjing, China). The modified Lowry protein assay kit (Pierce, Rockford, IL, United States) was used to quantify protein. Equal amounts of protein were analyzed using 10% SDS–PAGE, and transferred to a PVDF membrane. PVDF membranes were incubated with primary rabbit anti-LC3B (CST, 3868S,1:1200), primary mouse anti-cathepsin B (ab58802,1:1000), rabbit anti-P53 (ab131442,1:1000), rabbit anti-p21Cip1p (CST,2947S,1:1000), rabbit anti-p16INKa (CST,80772S,1:1000), rabbit anti-P62 (CST,16177S,1:1000), and anti-GAPDH (ab181602,1:2000) incubated overnight at 4°C. Following subsequent incubation with anti-rabbit HRP-conjugated secondary antibody (Affinity, S0001, 1:2000) or anti-mouse HRP-conjugated secondary antibody (Affinity, S0002, 1:2000), blots were visualized with enhance chemiluminescence (Millipore, Billerica, MA, United States). Bands on X-ray film were scanned with GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, United States). The intensity of bands was quantified with Quantity one software. All values were normalized to the corresponding GAPDH.

Cells were lysed in RIPA cell lysis buffer supplemented with a protease inhibitor mixture, followed by centrifugation and supernatant collection. Following SDS-PAGE, the proteins were transferred onto PVDF membranes (Millipore, Billerica, USA) which were subsequently blocked. Next, the membranes were incubated with primary anti-C5AR1 (1:500; MA5-16937; Invitrogen) or anti-GAPDH (1:3000; AF7021; Affinity, Changzhou, China) antibodies at 4°C overnight, followed by incubation with HRP-labelled secondary antibody (1:3000; S0001; Affinity) for 1 h at room temperature. The protein bands were developed using an Enhanced chemiluminescence (ECL) luminous substrate (P90719; Millipore) and photographed with a chemiluminescence imaging system (ChemiScop Series 3600; Enco Biology, Shenzhen, China).

Kidney tissues or cells were lysed and subsequently sonicated in PBS that contained 1% Triton X-100, 250 mmol/L phenylmethanesulfonyl fluoride, 2 mmol/L EDTA, and 5 mmol/L dithiothrietol (pH7.5). The total protein concentration was then determined by the BCA protein assay reagent kit (GBC, G3422). Thirty milligrams of protein for each sample was denatured in boiling water for 5 min then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were blocked 1 h with 5% nonfat dry milk in Tris-buffered saline, followed by incubation overnight with rabbit α-smooth muscle actin (α-SMA) antibody (Affinity, AF1032), Collagen IA (Affinity, AF7001), NLRP3 (Abcam, ab214185), Caspase-1 (Affinity, AF4005), APJ (Fitzgerald, 70R-51439), p47phox (Affinity, AF5220) or p22phox (Affinity, DF10099) at 4 °C. For β-actin, the membranes were stripped and reprobed with rabbit anti-β-actin antibody (Affinity, AF7018) or anti-GAPDH antibody (Affinity, AF7021). After being washed with Tris-buffered saline, membranes were incubated with a secondary antibody labeled with horseradish peroxidase-conjugated secondary antibody (Affinity, S0001) and visualized using enhanced chemiluminescence. The intensities of blotted bands were quantified with the software (ImageJ, free download from http://rsbweb.nih.gov/ij/).

Total protein from samples was extracted using RIPA buffer (Cwbio, Beijing, China). The protein concentration was then quantified using the BCA Protein Assay Kit (Cwbio, Beijing, China) and diluted accordingly. Equal amounts of protein were separated via SDS-PAGE with 8% or 10% polyacrylamide gels, depending on the molecular weight of the protein. The separated proteins were then transferred to a 0.45 μm PVDF membrane (Millipore,St. Louis, MO, USA) via electrophoresis, followed by blocking with 5% BSA at room temperature for 2 h. Primary antibodies for each protein were diluted in 5% BSA as follows: Fibronectin (1:1000, 26836S, CST), Col-III (1:1000, 66887S, CST), VASH-1 (1:1000, sc-365541, Santa Cruz), p-Smad3 (1:1000, 9520S, CST), Smad3 (1:1000, 9513S, CST), β-actin (1:1000, #AF7018, Affinity), and EGFP (1:5000, SRP15324, Saier); the secondary antibodies used were anti-rabbit (1:40,000, S0001, Affinity) and anti-mouse (1:40,000, S0002, Affinity). All primary antibodies were incubated overnight at 4 °C, followed by 1.5 h of incubation at room temperature with the corresponding secondary antibodies. Detection was performed using the ECL chemiluminescence detection kit (Biosharp, Guangzhou, China). Image collection and quantitative analysis were conducted using ImageJ software v1.8.0.