Ab51132

The testis tissue was lysed on ice with RIPA buffer as previously described [45 (link), 50 (link)]. Nuclear proteins were extracted by NE-PER™ Nuclear Extraction Reagents (Thermo Scientific). The protein concentrations were measured by a BCA Protein Assay Kit. The proteins were loaded into 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk for 1 h and incubated with primary antibodies at 4 °C overnight and with secondary antibodies at room temperature for 1 h. Primary antibodies against NLRP3 (ab270449, 1:1000), ASC (ab151700, 1:1000), GAPDH (ab8254, 1:1000), ZO-1 (ab216880, 1:500), occludin (ab216327, 1:500), NF-κB (ab32536, 1:1000), proliferating cell nuclear antigen (PCNA, ab29), phospho-p47phox (ab63554, 1:1000), p47phox (ab166930), and Cab39 (ab51132, 1:1000) were purchased from Abcam (Cambridge, UK). Phospho-ACC (1:1000 dilution, #3661S), ACC (1:1000 dilution, #3676), phosphor-AMPK (1:1000 dilution, #2535), and AMPK (1:1000 dilution, #2603) were purchased from Cell Signalling Technology. The protein bands were detected using ECL (Bio-Rad, USA) and normalized to GAPDH.

Paraffin sections prepared from in vivo experiments were used for immunohistochemistry assays to detect protein expression levels of CAB39, p-AMPK and p-mTOR expression level. The indirect streptavidin-peroxidase method was used according to the manufacturer’s instructions. The antibodies used were rabbit anti-CAB39 (ab51132, Abcam), anti-p-mTOR (ab109268, Abcam) and anti-p-AMPK (orb5692, Biorbyt).

Sodium palmitate (#P9767) and Oil Red O (ORO, #O0625) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Dimethyl sulfoxide (DMSO, #D8371), fatty acid free bovine serum albumin (BSA, #A8850) and Nile red (#N8440) was obtained from Solarbio (Beijing, China). Radicicol (#HY-N6769) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). The primary antibodies against LKB1(3047s, 1:1000), phospho-LKB1^Ser428 (3482s, 1:1000), AMPKα (2732s, 1:1000), phospho-AMPKα^Thr172 (2535s, 1:1000), ACC (3676s, 1:1000), phospho-ACC^Ser79 (11818s, 1:1000), FASN (3180s, 1:1000) and β-actin (4970s, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for CPT1α (ab128568, 1:1000), MO25 (ab51132, 1:10000) and STRAD (ab192879, 1:2000) were bought from Abcam (Cambridge, MA, USA). Antibodies against LKB1(10746-1-AP) were obtained from Proteintech (Rosemont, IL, USA). Anti-SREBP1 (sc-13551, 1:200) and anti-Nitro-tyrosine (sc-32757) were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, Texas, USA). Alexa Fluor^® 488 conjugated WGA (#W11261) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit Control IgG (#AC005) and Mouse Anti-Rabbit IgG Light Chain (#AS061, 1:5000) were purchased from ABclonal Biotechnology (Wuhan, China).