Anti ha antibody c29f4

Cellular proteins were extracted, separated by gel electrophoresis, transferred to nitrocellulose membranes and blocked with 5% nonfat dry milk as previously described [27 ]. The blots were then incubated overnight at 4 °C with primary antibodies, as follows: rabbit monoclonal anti-HA antibody (C29F4; Cell signaling) 1:5000 dilution, rabbit anti-GAPDH antibody (14C10; Cell Signaling, Danvers, USA) or rabbit polyclonal anti-AdipoR2 (1:1000, described in [44 (link)]). Blots were then washed with PBS-T and incubated with a swine anti-rabbit IgG/HRP (1:3000 dilution; Dako, Glostrup, Denmark) or goat anti-mouse HRP (1:3000, Dako, Glostrup, Denmark) then washed again with PBS-T. Blots were developed using an ECL kit (Immobilon Western; Millipore), and the signals documented using a digital camera (VersaDoc; Bio-Rad, Hercules, USA). The membranes were then stripped and reprobed with anti-GAPDH (14C10) rabbit IgG (1:2500 dilution; Cell Signaling, Danvers, USA), which served as a loading control. The PageRuler Plus prestained protein ladder was used to assess molecular weight (Thermo Fisher Scientific, Carlsbad, USA).

Cells transfected or infected with control or NANEP-expressing vectors were lysed with RIPA buffer (50 mM Tris pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with fresh protease inhibitors for 20 minutes on ice. Proteins were then analyzed on SDS-PAGE electrophoresis followed by Western Blot using specific primary antibodies anti-flag antibody: F3165 from Sigma (1:1000), anti-HA antibody C29F4 from Cell Signaling (1:1000), anti-GAPDH: 2118S from Cell Signaling (1:1000), GFP NB1001770 from Novusbio (1:1000) and appropriate HRP secondary antibodies (HRP anti mouse 170-65-16 (1:3000), HRP anti rabbit 170-6515 (1:3000), and HRP anti-goat 1721034 (1:5000) from BioRad). Chemiluminescence was determined using the ECL detection kit (GE Healthcare Amersham) and band intensities were quantified using Image Studio Lite software.

For immunofluorescence, fixed hearts were washed three times for 30 min in PBS and then fixed with 4% PFA for 2 h before submerging in 30% sucrose. Hearts were subsequently embedded in OCT and frozen on dry ice. The frozen block was then mounted on a Leica microtome (Leica biosystems, UK) and tissue sections of 10 μm were collected onto glass microscope slides. Tissues were subsequently stained using 1:400 dilution of primary antibody in PBS plus 0.4% Triton X-100 and 10% donkey serum. Antibodies used: SERCA2a (MA3-910, Thermo Fisher Scientific, USA) and anti-HA antibody C29F4 (Cell Signalling Technology, USA). The tissues were subsequently washed with blocking buffer and incubated with fluorescently labelled secondary antibodies. Nuclei were stained with Gold Anti-fade Reagent with DAPI (Invitrogen, USA). A confocal microscope (ZEISS, LSM 700) was used for imaging analysis.