Anti cx43

Immunofluorescence was performed as described previously [27 (link)]. Ishikawa cells were grown on glass coverslips and transfected with GPR64 siRNA. Upon completion of growth, coverslips were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% of Triton X-100 (Sigma-Aldrich, St. Louis, MO). After further washing, Ishikawa cells were exposed to anti-GPR64 (sc-69,492; Santa Cruz Biotechnology) and anti-Cx43 (#3512; Cell Signaling) antibodes overnight at 4 °C and secondary antibodes for 2 h at room temperature. Washed coverslips were then mounted onto microscope slides with a DAPI-impregnated mounting media (Vector Laboratories) to enable nuclear visualization, and images were captured via a Zeiss LSM700 confocal microscope (Carl Zeiss microImaging GmBH, Jena, Germany) using ZEN 2009 software (Carl Zeiss microImaging).

The primary antibodies were mouse monoclonal anti-prion protein (anti-PrP, clone SAF83 for mouse and SAF54 for human samples, 1:100; Bertin Pharma Co.), anti-α-actinin (anti-ACTN, A7811, clone EA-53, 1:400; Sigma-Aldrich) and anti-hypoxia inducible factor-1 α (anti-HIF-1α, MA1–516, clone mgc3, 1:200; Pierce-Thermo), rabbit polyclonal anti-connexin 43 (anti-Cx43, #3512, 1:100; Cell Signaling Technology) and anti-connexin 45 (anti-Cx45, #sc-25716, 1:50; Santa Cruz Biotechnology), and goat polyclonal anti-cardiac troponin T (anti-cTnT, sc-8121, 1:50; Santa Cruz Biotechnology) antibodies.

The total protein was isolated by lysing frozen LV tissue in RIPA buffer supplemented with 10 µM 1,4-dithiothreitol (Sigma-Aldrich) and protease and a phosphatase inhibitor cocktail (Roche Diagnostics) on ice. For SDS-PAGE 50 µg protein was loaded and transferred to a nitrocellulose membrane after separation. The following primary antibodies were used: anti β-GAL (Cell Signaling #27198), anti-CD44 (Abcam ab157107), anti-CX43 (Cell Signaling 3512), anti-iNOS (Cell Signaling #13120), anti-MHC (Abcam ab50967), anti-p-p65^S536 (Cell Signaling #3033), anti-p65 (Cell Signaling #8284), anti-PPCA (Abcam ab184553), anti-NEU1 (Santa Cruz Biotechnology sc-32936), anti-NEU3 (Santa Cruz sc-134931). Chemiluminescence detection was carried out after incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare) and enhanced chemiluminescence reagents (PerkinElmer) using the ChemiDoc™ MP system (Bio-Rad). Image LabV5.0 software (Bio-Rad) was used for quantification and Ponceau S staining served as a loading control.

Immunofluorescence was performed as previously described [43 (link)–46 (link)]. Briefly, samples were incubated overnight at 4 °C with mouse monoclonal anti-GFAP (BD Biosciences, Cat# 610566, RRID: AB_397916, 1:500), rabbit polyclonal anti-Cx43 (Cell Signaling Technology, Cat# 3512, RRID: AB_2294590, 1:200), goat polyclonal anti-IBA1 (Novus Biologicals, Cat# NB100-1028, RRID: AB_521594, 1:500). The following day, sections were washed in 0.1% Triton X-100 in PBS 3 times at room temperature and then incubated 1 hr at room temperature with appropriate combination of secondary antibodies: goat polyclonal anti-mouse (Alexa Fluor 488, Thermo Fisher Scientific, Cat# A-11001, RRID: AB_2534069, 1:1’000), goat polyclonal anti-rabbit (Alexa Fluor 564, Molecular Probes, Cat# A-11010, RRID: AB_143156, 1:1’000) and donkey anti-goat (Alexa Fluor 647, Thermo Fisher Scientific, Cat# A-21447, RRID:AB_2535864). Nuclei were counterstained with DAPI (1:10’000, Invitrogen) for 5 min at room temperature and then mounted with BrightMount mounting medium (Abcam). Profile plots for immunofluorescence images were obtained as previously described [43 (link)].

Cells were collected using 0.05% Trypsin-EDTA (Thermo Scientific, Waltham, MA, USA). After phosphate-buffered saline (PBS) rinsing, cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Jiangsu, China). Protein concentration was measured using a BCA Protein Assay kit (Beyotime). Equivalent quantities of protein (30 µg) were injected and then loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by the transferring of gels onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). After that, the membranes were blocked in 5% non-fat milk for 1 h at room temperature and then incubated (4°C, overnight) with primary antibodies anti-Cx43 (#3512, Cell Signaling Technology, Danvers, MA, USA) and Tubulin (11224-1-AP, Proteintech, USA). Following 1-h incubation (room temperature) with horseradish-peroxidase (HRP)-conjugated secondary antibodies. Finally, the blots were visualized using Super Enhanced Chemiluminescence (ECL) Detection Reagent (Beyotime), and the signals were analyzed using ImageJ software. Chemiluminescence was determined using a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China).

Western blot analyses were performed as described previously [27 (link)]. Briefly, twenty five micrograms of protein lysates were electrophoresed via SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was blocked with Casein (0.5% v/v) prior to exposure to anti-GPR64 (sc-69,492 Santa Cruz Biotechnology), anti-phospho-AMPK (Thr 172, #5235; Cell Signaling, Danvers, MA), anti-AMPK (#2532, Cell Signaling), anti-Cx43 (#3512, Cell Signaling), and anti-β-actin (SC-47778; Santa Cruz Biotechnology) antibodies. Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked secondary antibody followed by exposure to electrochemiluminescence reagents (ECL) according to the manufacturer’s instructions (GE Healthcare Biosciences, Piscataway, NJ).