Af7001

We established a tissue microarray (TMA, 3 mm) consisting of LUAD samples: 14 AIS samples, 18 MIA samples, and 17 IAC samples (Supplementary Table 4). After antigen repair and blocking, the TMA was incubated with specific primary antibodies (EPCAM, Servicebio, GB14078, 1:200; FOXP3, Servicebio, GB11093, 1:200; TPSB2, Novus, NBP2-33551, 1:300; CD79A, Novus, NB100-64347ss, 1:200; CLDN5, Servicebio, GB11290, 1:200; COL1A1, Affinity, AF7001, 1:200; UBE2C, Abcam, ab12290, 1:50; p-SMAD2, Affinity, AF8314, 1:200; SCGB1A1, Servicebio, GB111412, 1:200; PDPN, Affinity, AF3670-SP, 1:200; FCN1, Novus, NBP1-84706, 1:200; GHD, Proteintech, 67538 1:250; PCNA, Servicebio, GB11010, 1:300), incubated with horseradish peroxidase (HRP)-labeled secondary antibodies or fluorophore-labeled secondary antibodies, and finally stained with diaminobenzidine and counterstained with hematoxylin or stained with DAPI. Three independent pathologists distinguished the pathological type for the TMA.

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/).

RIPA buffer (Beyotime Bio., Shanghai, China) was used to prepare cell extracts for immunoblotting. Afterwards, total protein content was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Protein samples were electroblotted onto polyvinylidene difluoride (PVDF) membranes. TBST (Tris-buffered saline containing Tween 20) buffer with 5% bovine serum albumin was used to block the membranes. The membranes were blocked at room temperature for 1 h, and then, the corresponding primary antibodies, α-SMA (Affinity Biosciences, Cincinnati, OH, USA; AF1032; 1 : 500), COL1A1 (Affinity Biosciences; AF7001; 1 : 500), TGF-β (Abcam, Cambridge, MA, USA; Ab179695; 1 : 3000), FGFR4 (Affinity Biosciences; DF10316; 1 : 1000), and GAPDH (Cell Signaling Technology, Danvers, MA, USA; #5174; 1 : 2000), in TBST buffer were used to incubate the membranes. The membranes were kept at 4°C overnight. On the next day, the membranes were incubated with secondary antibodies (Beyotime) at room temperature for 1 h. An enhanced chemiluminescence chromogenic substrate (Thermo Fisher Scientific) was used for visualized protein bands.