Mouse anti myc

Cells were seeded in 48-well plates in triplicate overnight to reach 70–90% confluency. Cultures were transfected with 0.2 µg DNA/well using Lipofectamine™ 3000 (Invitrogen) following the supplier’s protocols. 48 h later, cells were fixed in 4% PFA for 10 min, treated with 0.3% Triton-X for 5 min and blocked with Superblock (Thermo Fisher, Rockford, IL, USA) for 30 min at room temperature. Cells were then incubated with mouse anti-myc (Cell Signaling Technology, Danvers, MA, USA) for 1 h, washed with PBS and incubated with the secondary antibody for another hour. Cultures were stained with Hoechst 33342 to detect total cells and apoptotic nuclei. Images were acquired using a Zeiss epifluorescence microscope with a digital camera and Axiovision software. The proportion of cells with apoptotic nuclei was quantified by scoring all transfected cells from 4 random fields for each sample.

Cells were lysed in PBS that contained 1% Triton X-100 and a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Cell lysates were then cleared by centrifugation at 12,000× g at 4 °C for 15 min. Alternatively, cells were directly lysed in an electrophoresis sample buffer. For coimmunoprecipitation, GFP-trap and Myc-trap (ChromoTek GmbH, Munich, Germany) or Flag M2 conjugated magnetic beads (Sigma) were added, and the mixture was incubated at 4 °C for 2.5 h. After washing with PBS containing 1% Triton X-100, proteins bound to the beads were eluted by adding 2× electrophoresis sample buffer and analyzed by immunoblotting. The primary antibodies used in the study were as follows: Rabbit and mouse anti-Flag (Sigma Aldrich), mouse anti-Myc (Cell Signaling, Danvers, MA, USA), rabbit anti-Myc (MBL, Tokyo, Japan), mouse anti-GFP (Roche, Basel, Switzerland), mouse anti-GFP (Santa Cruz, Dallas, TX, USA), rabbit anti-NS1.2, anti-NTPase, and anti-NS4. Rabbit anti-NS1.2 was produced in this study immunized rabbit with NS1.2 (1–202), which was purified from bacteria expressed His-tagged NS1.2 (1–202) (AllBio Science Incorporated, Taichung Taiwan). Anti-NTPase and NS4 were kindly provided by Dr. Pey-Jium Chang [33 (link)].

Embryos were fixed in 4% PFA, rinsed in PBT, digested briefly in 10 μg/mL proteinase K, refixed in 4% PFA, rinsed in PBT, and blocked in 2 mg/mL BSA + 2% goat serum in PBT. Embryos were then incubated overnight in rabbit anti-Laminin (Sigma L9393) at 1:200, mouse anti-Myc (Cell Signaling 2276) at 1:1000, or rabbit anti-phospho histone H3 (Upstate 06-570) at 1:500 in blocking solution, rinsed in PBT, and incubated overnight in Alexa Fluor 488 anti-Rabbit IgG, 568 anti-Rabbit, or 568 anti-Mouse (Invitrogen) at 1:1000 in PBT. Embryos were co-stained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride and rinsed in PBT prior to mounting in agarose for confocal imaging.

HEK293T cells expressing wild type and mutant PKCγ were treated with PMA (20 nM, 10 min) (Sigma Aldrich, St. Louis, MO, United States) to obtain PKCγ translocation. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and washed three times with PBS. With the exception of PKCγ-EGFP transfected cells, cells were permeabilized and blocked in 0.1% Triton™ X-100, 5% fetal bovine serum in PBS for 30 min at room temperature, followed by incubation overnight at 4°C with primary antibody mouse anti-Myc (Cell Signaling, Danvers, MA, United States; 1:100). The next day, the cells were washed with PBS and incubated with secondary antibody including mouse anti-Alexa Fluor® 488 (Santa Cruz Biotechnology, TX, United States; both 1:500) for 1 h at room temperature in phosphate buffer. All slides were mounted in Vectashield medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, CA, United States). Images were obtained with structured illumination microscopy and processed using ImageJ software (http://fiji.sc/, National Institutes of Health, Bethesda, MD, United States).

The mouse polyclonal antibody against CP204L and rabbit polyclonal antibody against B646L were prepared in our laboratory. Polyclonal rabbit anti-SNX32 (catalog no. 25763-1-AP), rabbit anti-p62 (catalog no. 18420-1-AP), and rabbit anti-RAB1B (catalog no. 17824-1-AP) were purchased from Proteintech. Rabbit anti-LC3B (catalog no. 3868s) and mouse anti-Myc (catalog no. 2276S) were purchased from Cell Signaling Technology. Monoclonal mouse anti-HA (catalog no. H3663), mouse IgG polyclonal antibody (catalog no. 12-371), rabbit IgG polyclonal antibody (catalog no. 12-370), and mouse anti-Flag (catalog no. F1804) were purchased from Sigma-Aldrich. Mouse anti-β-actin was purchased from Santa Cruz (catalog no. sc-58673). Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 4408s) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 8889s) antibodies were purchased from Cell Signaling Technology; IPKine goat anti-mouse IgG heavy chain secondary antibody, HRP labeling (elimination of light chain interference) (catalog no. A25112) and IPKine goat anti-mouse IgG light chain secondary antibody, HRP labeling (elimination of heavy chain interference) (catalog no. A25012) were purchased from Abbkine. Dimethyl sulfoxide, 3-MA (autophagosome formation inhibitor), MG132 (proteasome inhibitor), and NH₄Cl (lysosome inhibitor) were purchased from Sigma-Aldrich.

For plaques, immunostaining, and PLA, primary antibodies used were mouse anti-RSV N (Novus Biologics, Cat. No. NB100-64752 1:500 dilution), human anti-RSV F (Palivizumab, Medimmune, 1 μg/mL), mouse anti-RSV P (Abcam, Cat. No. ab94965, 1:1000 dilution), mouse anti-RSV M2-1 (Abcam, Cat, No. ab94805, 1:1000 dilution), goat anti-panRSV (Abcam, Cat. No. ab20745, 1:1000 dilution), rabbit anti-FLAG (Cell Signaling Technology, Cat. No. 14793S, 1:1000 dilution), mouse anti-V5 (Sigma Aldrich, Cat. No. V8012, 1:500 dilution), mouse anti-Myc (Cell Signaling Technology, Cat. No. 2276S, 1:1000 dilution), mouse anti-Myc-AF488 (Cell Signaling Technology, Cat. No. 2279S, 1:250 dilution), rabbit anti-HA (Santa Cruz, Cat. No. sc-805, 1:1000 dilution), and mouse anti-HA-AF488 (Novus Biologics, Cat. No. NBP2-50416AF488, 1:100 dilution). All respective secondary antibodies for immunostaining (Jackson Immunoresearch, Thermo Fisher) were used at 4 μg/ml. Secondary antibodies for PLA (Sigma Aldrich) were used according to manufacturer’s instructions.