Mouse anti ha antibody

The muscle samples were fixed in a 4% formaldehyde solution at room temperature for 30 min. Subsequently, the samples were rinsed with PBST (PBS with 0.1% Triton X-100) for 4 times, 15 min each time. The samples were then treated with a blocking solution containing 5% BSA in PBS. Following this, the samples were incubated with primary antibodies overnight at 4 °C. Afterwards, the samples were washed again with PBST for 4 times, 15 min each time, and then incubated with secondary antibodies labeled with Alexa Fluor 568 or Alexa Fluor 488 (1:500 dilution, Invitrogen) for 2 h at room temperature. After washing with PBST for 4 times, 15 min each time, the samples were mounted using Slow-Fade mounting medium (Invitrogen) for imaging. The primary antibodies used in this study were chicken anti-GFP antibody (1:1000, Abcam, ab13970); mouse anti-HA antibody (1:1000, Santa Cruz, sc7392); mouse anti-Cnx99A antibody (1:100, DSHB). Representative images were shown for each genotype, with eight individual samples for each genotype analyzed. All tissue immunostaining images were obtained under identical gain settings as well as laser intensities.

Antibodies against CD8 and rabbit HA were from Sigma, antibodies against BAP31, LAMP1, actin, Hsp70 and β-tubulin were from AbCam, anti-ERGIC53 antibody was from Alexis, mouse anti-HA antibody was from Santa Cruz Biotechnology, anti-BiP antibody was from Cell Signaling, anti-CNX and -CRT antibodies for immunoblotting were from Stressgen, anti-CRT antibody for immunofluorescence was from Thermo Scientific and anti-GM130 antibody was from BD Biosciences. Antibodies against opsin and STT3B antibodies were provided by Stephen High (University of Manchester, Manchester, UK). IRDye 800 CW and IRDye 680 RD were from LI-COR, and secondary antibodies for microscopy were from Jackson Laboratories (Stratech Scientific). The inhibitors leupeptin (Enzo Life Sciences), pepstatin A (Sigma), Z-LLF-CHO (PSII, Calbiochem), chloroquine (Sigma) and cycloheximide (CHX, Sigma) were used at 0.5 mM, 1 µg/ml, 10 µM, 5 mM and 100 µg/ml, respectively.

Rabbit anti-MAVS antibody was obtained from Bethyl Laboratories. Mouse anti-HA antibody, mouse anti-V5, goat anti-EEA1 and rabbit anti-GAPDH HRP-conjugated antibodies were purchase from Santa Cruz Biotechnology. Mouse anti-VSVG (P5D4) and mouse anti-DENV (clone D3-2H2-9-21) antibodies were purchased from Santa Cruz Biotechnology and Millipore, respectively. Alexa Fluor 488 or 594 phalloidin and Alexa fluor-conjugated secondary antibodies were purchased from Invitrogen.

The following primary antibodies were used: mouse anti-α-acetylated-tubulin antibody (Santa Cruz Biotechnology; 1:1000), mouse anti-Islet-1 antibody (DSHB; 1:200), mouse anti-Zn-8 antibody (ZIRC; 1:200), rabbit anti-glial fibrillary acidic protein (Sigma; 1:500), mouse anti-Zpr-1 antibody (ZIRC; 1:200), mouse anti-HA antibody (Santa Cruz Biotechnology; 1:100), and mouse anti-GFP antibody (Molecular Probes; 1:200).

Cells (1 × 10⁶) were washed twice with PBS (phosphate buffered saline), lysed in 500 μL of 2× SDS-PAGE sample buffer (8% SDS, 40% glycerol, 0.25 M Tris-HCl (pH 6.8), 2% BPB and 5% β-mercaptoethanol), and boiled for 10 min. Following SDS-PAGE, the gel was subjected to western blotting and visualized using a LAS3000 mini (Fujifilm Co., Tokyo, Japan) and Immobilon Western Reagents (Millipore). Antibodies used in the experiments were as follows: mouse monoclonal (M2) anti-FLAG antibody (dilution; 1:5000) was purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA); the rabbit polyclonal anti-Myc antibody (1:5000), the mouse anti-GST antibody (1:5000), and the mouse anti-HA antibody (1:5000) were obtained from Santa Cruz biotechnology; the mouse anti-(His)₆ antibody (1:500) was obtained from Roche; the mouse anti-GAPDH antibody (1:5000) was from Millipore. Secondary antibodies conjugated with horseradish peroxidase (anti-Rabbit IgG antibody (1:5000) and anti-Mouse IgG antibody (1:5000, except for the case with anti-GAPDH (1:10,000))) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).