Anti gapdh

The following antibodies were used for Western blotting and immunofluorescence analyses: anti-GAPDH (CWBIO, Beijing, China, CW0100 M; WB, 1:1000), anti-Acta2 (HUABIO, Hangzhou, China, ER1003; IF, 1:100; WB, 1:1000), anti-Myh11 (Proteintech, Wuhan, China, 60222-1-lg; IF, 1:100; WB, 1:5000), anti-PCNA (HUABIO, Hangzhou, China, ET1605-38; IF, 1:100; WB, 1:1000), anti-MMP2 (Thermo Fisher Scientific, Shanghai, China, 436000; IF, 1:100; WB, 1:1000), anti-4-HNE (R & D Systems, Shanghai, China, MAB3249; IF, 1:50; WB, 1:1000), anti-Nrf2 (Abcam, Cambridge, UK, ab31163; IF, 1:100; WB, 1:1000), anti-TNF-α (Santa Cruz Biotechnologies Inc., CA, USA, sc-52746; IF, 1:200; WB, 1:1000), anti-IL-1β (Santa Cruz Biotechnologies Inc., CA, USA, sc-515598; IF, 1:200; WB, 1:1000), anti-PI3K (CST, Shanghai, China, #4249; WB, 1:1000), anti-p-PI3K (CST, Shanghai, China, #17366; WB, 1:1000), anti-Akt (CST, Shanghai, China, #4685; WB, 1:1000), anti-p-Akt (CST, Shanghai, China, #4060; WB, 1:1000).

Frozen lung tissues were weighed (100 mg) and homogenized in 1,000 μl RIPA buffer containing protease inhibitors (CW Biotech, Beijing, China). Lysates were centrifuged at 12,000 rpm for 20 min at 4°C and the protein concentration of the supernatant was quantified by BCA assays (Beyotime, Shanghai, China). Samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, United States) by the wet transfer method. Membranes were blocked with 5% skim milk solution and incubated with anti-p-AMPKα (Thr¹⁷²), anti-AMPKα at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, United States) and anti-GAPDH at 1:2,500 dilution (CW Biotech) overnight at 4°C. The corresponding HRP-conjugated anti-rabbit IgG or anti-mouse IgG was incubated at room temperature for 1 h to detect primary antibodies. Blots were visualized with enhanced chemiluminescence reagents (Millipore) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Transgenic flies expressing the human TDP-43 (Wt or A315T-mutant) were described previously [40 (link),41 (link),87 (link)]. GMR-Gal4, OK371-Gal4, Elav-Gal4 and UAS-Lon-RNAi lines were obtained from the Bloomington Drosophila Stock Center (BDSC). Another UAS-Lon-RNAi fly line was obtained from the Vienna Drosophila Resource Center (VDRC). UAS-dLonOE was from the Kyoto Stock Center. The Tubulin-Gal80^ts (Tub-Gal80^ts) line was kindly provided by Dr. A. Guo (IBP, CAS) [48 (link)]. The UAS-mito-roGFP2-Grx1 fly lines were kindly provided by Dr. T. Dick [47 (link)].
For flies under the Elav-Gal4/Tub-Gal80^ts-driver or GMR-Gal4/Tub-Gal80^ts-driver, parental flies were crossed and cultured at 18°C, young flies after eclosion were transferred to 28°C for 4 hr every day to induce TDP-43 expression. Other flies were all cultured at 25°C. All flies were raised in standard fly food, 50% relative humidity, and 12hr-12hr light-dark cycles as described previously [41 (link),70 (link),87 (link)].
Antibodies used in this study include polyclonal rabbit-antibodies against TDP-43, ATP5A1, LonP1, HSPA9, ClpP, TOM20 and IMMT (ProteinTech Group Inc), as well as mouse monoclonal antibodies, anti-actin (ProteinTech Group Inc), anti-HSP60 (BD Biosciences) and anti-GAPDH (CWBIO). Rat-anti-dElav antibody is a kind gift from Dr. A. Guo.

Cells and tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Vazyme). The protein concentration of cell lysates was quantified by a BCA Protein Assay kit (Cwbio). The primary antibodies were anti-PD-L1 (1:1000, CST), anti-c-Myc (1:1000, CST), anti-p-S6 (1:1000, CST), anti-S6 (1: 1 000, CST), anti-p-STAT3 (1:1000, CST), anti-STAT3 (1:1000, CST), anti-p-AKT (1:1000, CST), anti-AKT (1:1000, CST), and anti-GAPDH (1:2000, Cwbio). HRP-conjugated secondary antibody (1:2000, CST) and ECL reagent (Millipore) were used for detection of immunoreactive proteins captured by ImageQuant LAS 4000 detection system.

Primary antibodies: Anti-Sox3 (GeneTex, GTX132494) was purchased from GeneTex, Irvine, California, USA. Anti-caspase3 (H-277) was from ZSGB-BIO, Beijing, China. Anti-Gapdh (glyceraldehyde-3-phosphate dehydrogenase, CW0100) was purchased from CWBIO, Beijing, China. Anti-Cyp19a1 (A12684) was from ABclonal, Wuhan, China.
Secondary antibodies: goat anti-rabbit IgG (H + L), horseradish peroxidase (HRP) conjugated antibody (31460) and goat anti-mouse IgG (H + L), HRP conjugated antibody (31430) were purchased from Invitrogen, Carlsbad, USA. FITC-conjugated immunopure goat anti-rabbit IgG (H + L) (ZF-0311) was from Feiyi Technology, Wuhan, China.

Full-length NAIP5 and NAIP2 genes were obtained by DNA synthesis. Mouse NLRC4, pro IL-1β and pro caspase-1 were amplified from reverse-transcribed mouse cDNAs. Full-length PrgJ and FliC genes were amplified from S. typhimurium genomic DNA. NLRC4-R288A-L435D-1 008-1 012^DDYD−AAAA(NLRC4^M), all NAIP5 and flagellin mutations were generated by standard molecular biology procedures. FliC_D0_L was designed as described before^{32 (link)}. All constructs were verified by sequencing.
Antibody used: anti-Myc (cw0299, CWBIO), anti-HA (cw0092, CWBIO), anti-IL-1β (GTX74034, GeneTex) and anti-GAPDH (cw0100, CWBIO).