Sc 7020

Cells were washed with PBS and lysed with Laemmli’s buffer, sonicated, and separated on SDS–PAGE gels followed by electro-transfer to nitrocellulose membranes. Antibodies used for western blot analysis were: anti-CagA (1:2000, sc-25766, Santa Cruz Biotechnology), anti-P-Tyr (1:1000, sc-7020, Santa Cruz Biotechnology), anti-GKN1 (1:500, HPA047684, Atlas antibodies), anti-GKN2 (1:11,000, ab188866, Abcam), anti-MUC5AC (1:1000, MA5-12178, Thermo Fisher Scientific), anti-MUC6 (1:500, AM10120SU-N, Acris), anti-α-tubulin (1:3000, T6074, Sigma-Aldrich), anti-β-actin (1:3000, A2228, Sigma-Aldrich), anti-mouse HRP (1:10,000; NA931, GE Healthcare), anti-rabbit HRP (1:10,000; NA934, GE Healthcare). HPR signals were detected using a solution of luminol (0.25 mg ml⁻¹, A8511-5G, Sigma-Aldrich) and p-coumaric acid (1.1 mg ml⁻¹, sc-215648A, Santa Cruz Biotechnologies) with an ImageQuant LAS 4000 CCD camera (GE Healthcare). Quantification of western blots was performed with ImageJ. Uncropped images of immunoblots are included as Source Data.

The smooth muscle layer of resistance PA rings was dissected. Total protein extracts and protein concentrations were prepared as previously described (2 (link)).
Immunoprecipitations were performed as follows: Lysates containing 0.5 mg total protein were incubated with 10 μg Kv1.2 (APC-010; Alomone Lab, Israel), Kv1.5 (APC-004), Kv2.1 (APC-012), Kv3.1b (APC-014), or Kv9.3 (HPA014864; Sigma, USA) monoclonal antibody overnight at 4°C, which were then captured by Protein A-Sepharose CL-4B beads (Santa Cruz Biotechnology, USA). The beads were collected by centrifugation at 200 g for 3 min at 4°C followed by 3 washes with lysis buffer. After the final wash, the immunoprecipitates were subjected to western blot analyses.
Immunoblotting was performed to detect phosphotyrosine levels in hypoxia-related Kv channels as described previously (24 (link)). Membranes were immunoblotted using antibodies to p-Tyr (SC-7020, Santa Cruz Biotechnology) at 4°C overnight. The signal intensity of the immunoreactive p-Tyr bands from the membrane protein was normalized to Kv channel expression. Fluorescent secondary antibodies (Rockland, USA) were visualized using an Odyssey scanner (LI-COR, USA).

Tamoxifen (T5648) was purchased from Sigma-Aldrich. Recombinant human NRG1-β1/HRG1-β1 (396-HB) and recombinant human Wnt3a (5036-WN) were from R&D Systems. mouse monoclonal antibody against β-catenin (610154) and rat monoclonal antibody against CD31 (PECAM1) (553370) were from BD Biosciences. Rabbit polyclonal antibody against GFP (ab6556) was from Abcam. TO-PRO^®-3 Iodide (642/661) (T3605) was from Invitrogen. Rabbit polyclonal antibody against ErbB2 (Neu) (C-18) (sc-284), rabbit polyclonal antibody against ErbB4 (C-18) (sc-283) and mouse monoclonal antibody against phosphorylated tyrosine (PY99) (sc-7020) were from Santa Cruz Biotechnology. Rabbit monoclonal antibody against GAPDH (14C10) (#2118) was from Cell Signaling Technology. Immunoprecipitation beads (Protein A Sepharose 4 Fast Flow (17-5280)) was from GE-Healthcare. Magnetic-activated cell sorting (MACS) beads, anti-rat IgG microbeads (130-048), were from Miltenyi Biotec. Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 were from Molecular Probes. Hypoxyprobe^TM-1 Omni Kit (HP3-100) was from Hypoxyprobe Inc.

We performed immunocytochemistry after electrical stimulation of human spermatozoa and washed and fixed them in cool methanol for 15 min at 4°C. Electrically stimulated spermatozoa were stained for intact acrosomal regions with pisum sativum agglutinin–fluorescein isothiocyanate dye (PSA–FITC conjugate: L0770, Sigma–Aldrich, US), and mitochondrial protein phosphorylation of sperm tail was detected using an anti-tyrosine phosphorylation antibody (anti-p-Tyr (pY99); SC-7020, Santa Cruz Biotech, USA). The primary antibody was an anti-pTyr(pY99) monoclonal mouse antibody in diluted phosphate buffer solution (PBS) with 0.01% bovine serum albumin (BSA), and was incubated 16hours at 4°C. The sample was then washed and treated for 1 h at room temperature with a secondary antibody, such as Cy5-labeled anti-mouse antibody with PSA–FITC (100 μg/mL). We finished the processing with nuclear count staining with 4’6’-diamidino-2-phenylindole (DAPI), and mounted the specimen with anti-fade solution (Vector shield® P100, USA) followed by imaging with a confocal microscope (ZISS M880, Germany). All images were processed and analysed for intensity of each red signal using the ZESS2016 software (ZEISS, Germany).

PASMCs were dispersed from 12-week-old rat resistance PAs. After enzymatic isolation, PASMCs were cultured at 4°C in Dulbecco's Modified Eagle's Medium (Corning, USA) supplemented with 15% fetal bovine serum for 2 h to allow cells to adhere to the plates.
Confocal microscopy imaging was performed with an Olympus confocal laser scanning microscope (BX61W1-FV1000, Japan). The Kv1.5 primary antibody (1:100; APC-150; Alomone Lab) and p-Tyr (1:100, SC-7020, Santa Cruz Biotechnology) were used as described previously. The secondary antibodies tetramethylrhodamine isothiocyanate (1:200; ZF-0316; ZSGB) and fluorescein isothiocyanate (1:150; A0568; Beyotime, China) were used. Nuclear staining was performed using 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 100 nM; Sigma) in fixed cells as previously described. Images of cells were analyzed using Image Pro Plus software version 5.0 (Media Cybernetics, USA).

Rapamycin (Rapa, R8781), 3-methyladenine (3-MA, M9281), bafilomycin A1 (Baf-A1, B1793), antibodies against ATG12 (WH0009140M1), MAP1LC3B (L7543), and ATG5 (WH0009474M1) were purchased from Sigma-Aldrich (Shanghai, CHINA), and MK-2206 2HCL (S1078) purchased from Selleckchem (Houston, TX, USA). Antibodies against Akt (9272), mTOR (2972), AMPK (2532), phospho-Akt (Ser473) (4060), LAMP1 (9091), phospho-mTOR (Ser2448) (5536), phospho-S6 ribosomal protein (Ser235/236) (2211), ribosomal protein (2217), phospho-c-Met (Y1234/Y1235) (4033), and c-Met (4560) were obtained from Cell Signaling Technology (Beverly, MA, USA), whereas antibodies against β-actin (sc-10731), VacA (sc-25790), CagA (sc-17450), phospho-tyrosine (PY99) (sc-7020) and SQSTM1 (sc-28359), and siRNAs specific for SQSTM1 (human, sc-29679), ATG12 (human, sc-72578), c-Met (human, sc-29397), and ATG5 (human, sc-41445), along with a control siRNA (sc-44230) were obtained from Santa Cruz Biotechnology (CA, USA).