Anti ha rat monoclonal antibody

The following antibodies were used: mouse monoclonal anti-β-actin (cat. no. A1978, 1:10,000; Sigma-Aldrich), rabbit polyclonal anti-FLAG (cat. no. 2368, 1:1000; Cell Signaling), mouse monoclonal anti-PARP-1 (sc8007, 1:1000; Santa Cruz), rabbit polyclonal anti-calnexin (sc11397, 1:1000; Santa Cruz), mouse monoclonal anti-V5 antibody (R960-25, 1:4000; Invitrogen), rat monoclonal anti-HA antibody (12158167001, 1:4000; Roche), rabbit polyclonal anti-HA antibody (71-5500, 1:1000 Invitrogen), goat polyclonal anti-calnexin (sc-6465, 1:200; Santa Cruz), sheep anti-TGN46 (AHP500, 1:200 AbD Serotec) and mouse monoclonal anti-GM130 antibody (610823, 1:200; BD Biosciences). Goat anti-mouse (cat. no. 31430, 1:5,000) and goat anti-rabbit IgG conjugated to horseradish peroxidase (31460, 1:5,000) were from Thermo Fischer Scientific. The following secondary antibodies were used for indirect immuno-fluorescence: FITC-conjugated donkey anti-sheep/goat (STAR88F, 1:200; AbD Serotec), Cy3-conjugated donkey anti-mouse IgG (715-165-150, 1:200; Jackson ImmunoResearch), Cy5-conjugated donkey anti-rabbit IgG (711-175-152, 1:200; Jackson ImmunoResearch), Cy2-conjugated donkey anti-mouse IgG (715-225-150, 1:200; Jackson ImmunoResearch), Cy5-conjugated donkey anti-goat IgG (705-175-147, 1:200; Jackson ImmunoResearch), Cy3-conjugated donkey anti-rabbit IgG (711-165-152, 1:200; Jackson ImmunoResearch).

Rabbit polyclonal anti-DYKDDDD antibody and mouse anti-GFP antibody were purchased from Cell Signaling Technology (Beverly, MA) and Nacalai Tesque (Kyoto, Japan), respectively. Rabbit polyclonal anti-Cullin2 (CUL2) and anti-TDP-43 antibodies were purchased from Abcam (Cambridge, UK) and Proteintech (Chicago, IL), respectively. Rat monoclonal anti-HA antibody was obtained from Roche (Basel, Switzerland). Mouse monoclonal antibodies against actin (C4), elongin B (D-5), and elongin C (56) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-FLAG antibody (M2) was obtained from Sigma. Rabbit anti-ubiquitin, Lys48-specific antibody was obtained from EMD Millipore (Billerica, MA). Mouse anti-Hsc70/Hsp70 antibody was purchased from StressGen (Victoria, BC). Mouse anti-HIF1α antibody was obtained from Abcam. Goat anti-mouse IgG Alexa fluor488 and fluor405 were purchased from Invitrogen (Carlsbad, CA), and goat anti-rabbit IgG CF568 was from BIOTIM (Hayward, CA). Mouse monoclonal anti-human VHL was generated as previously described31 (link).

Thin smears made from the synchronous culture of Pfcyp19b-OE lines were fixed using paraformaldehyde (Sigma) (4%) and glutaraldehyde (Sigma) (0.0075%) in 1xPBS (1^st Base) for 15 min, permeabilized in Triton X-100 (Sigma) (0.1%) for 10 min and then blocked with bovine serum albumin (BSA, Sigma) (3%) with glycine supplementation (1st Base) (22.52mg/ml) in PBS-T for 1 hour. Subsequently they were incubated with 1:200 rat monoclonal anti-HA antibody (Roche) and 1:200 rabbit polyclonal anti-PfBiP (BEI Resources) followed by incubation for 30 minutes with 1:2000 Alexa Fluor 594 goat highly cross-absorbed anti-rat (Jackson Immunoresearch) and 1:2000 Alexa Fluor 488 goat highly cross-absorbed anti-rabbit secondary antibodies (Jackson Immunoresearch). Nuclear counterstain was done with DAPI (Thermo Scientific) (1 ng/μl) for 10 min. All steps were performed at room temperature. Slides were mounted with ProLong Gold Antifade (Thermo Scientific). Images were captured with Zeiss LSM710 confocal microscope equipped with an Airyscan detector using a Plan-Apochromat 100x/1.46 oil objective and processed using Zen (Blue edition) imaging software (Zeiss). Images were analyzed using ImageJ. All antibodies have been tested for cross-reactivity and corresponding empty Vector control strain at the same generation and developmental stage was used parallelly as a negative control.

A549 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [0.15 mol/L sodium chloride, 1% Triton‐X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 5 mmol/L ethylene diamine tetraacetic acid (EDTA), 50 mmol/L Tris (pH 8)] (Sigma, EDTA from GE Healthcare, Buckinghamshire, UK, Tris from Merck Millipore, Darmstadt, Germany), supplemented with complete protease inhibitor (Roche, Mannheim, Germany). Protein concentrations were measured using the Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 15 µg protein was separated on NuPage Mini 3‐8% Tris‐Acetate gels (Invitrogen, Waltham, Massachusetts, USA) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). For probing of ABCA3‐HA, rat anti‐HA monoclonal antibody (Roche) and rabbit anti‐rat IgG (H + L) HRP secondary antibody (Southern Biotechs, Birmingham, AL) were used. β‐Actin (Santa Cruz, Dallas, TX) probing served as a loading control. SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for detection. Densitometric analysis was performed with Image J software.

All reagents and enzymes were from Sigma-Aldrich (St. Louis, MO, USA). Zymolyase was from ICN (Abingdon, UK) and Bacto Yeast Extract and Bacto Peptone were from Difco (Franklin Lakes, NJ, USA). Mitochondrial substrates were used as TRIS salts at pH 7.0. Solvents and salts used for HPLC were from J. T. Baker (Center Valley, PA, USA). Rat anti-HA monoclonal antibody and peroxidase-conjugated anti-rat IgG secondary antibody were obtained from Roche (Basel, Switzerland) and Jackson Immunoresearch (West Grove, PA, USA), respectively.

HEK293T cells were cotransfected with pNL4-3 plasmids harboring the CA mutations along with the pCEP4 mammalian expression vector, encoding HA-tagged human CypA, as described previously with slight modifications (40 (link)). Cells were cultured in the presence of 2 μM CsA when described. Reverse transcriptase-normalized viruses in 900 μl of culture medium were layered onto 500 μl of 20% sucrose in phosphate-buffered saline (PBS) and centrifuged at 20,000 × g for 2 h at 4°C. Pelleted virions were resuspended in 1× NuPAGE LDS sample buffer (Thermo) containing 2% β-mercaptoethanol, and the lysed virions were subjected to Western blotting. The HA-tagged CypA and p24 CA proteins were probed with a rat anti-HA monoclonal antibody (Roche Diagnostics) and mouse anti-p24 antibody (Abcam), respectively.

Pelleted cells were resuspended in 1 × NuPAGE LDS sample buffer (Thermo) containing 2% β-mercaptoethanol. Expression of endogenous MxB in IFN-β-treated cells was evaluated with Western blot using goat anti-MxB polyclonal antibody (SantaCruz Biotech) followed by HRP-conjugated donkey anti-goat IgG antibody (SantaCruz Biotech). Expression of HA-tagged host factors in SeV-infected MT4 cells was confirmed by Western blot using rat anti-HA monoclonal antibody (Roche Diagnostics) followed by HRP-conjugated goat anti-rat IgG antibody (American Qualex). The membranes were probed with rabbit anti-β-actin polyclonal antibody (Thermo) as a loading control. Chemiluminescence was detected using Chemi-Lumi One Ultra reagent (Nacalai Tesque) according to manufacturer's instructions.