Mouse anti flag

Primary antibodies: Mouse anti-Flag (Cell Signaling 8146), rabbit anti-V5 (Cell Signaling 13202), rabbit anti-His (Cell Signaling 12698), anti-Sf-SR-C-N polyclonal antibodies were generated by immunizing rabbits with purified GST-SR-C-N. Secondary antibodies: goat anti-mouse IgG-HRP conjugate (Santa Cruz sc-2005), goat anti-rabbit IgG-HRP conjugate (Cell Signaling 7074), rabbit anti-GST (Polyclonal, Bioss bs-2735R). The primary antibodies and secondary antibodies were used at 1: 1000 for western blotting. For immunostaining assays, Anti-V5-Dylight 488 conjugate (Invitrogen MA5-15253-D488, 1:200) and Alexa Fluor 488 goat anti-rabbit IgG (Cell Signaling 4412, 1:200) were used.
Inhibitors: Dynasore (dynamin inhibitor, TargetMol T1848, 7.5μM), chlorpromazine (clathrin-mediated endocytosis inhibitor, Millipore 215921, 40μM), monodansylcadaverine (clathrin-mediated endocytosis inhibitor, Sigma D4008, 150μM), nystatin (sequesters cholesterol, Millipore 475914, 20μM), cholesterol-oxidase (oxidize cholesterol, Millipore 228230, 4unit/ml), amiloride (macropinocytosis inhibitor, Millipore 129876, 150μM), Cytochalasin D (macropinocytosis inhibitor, Millipore 250225, 500nM), LY294002 (broad PI(3)K inhibitor, Cell Signaling 9901s, 50μM), and wortmannin (broad PI(3)K inhibitor, Cell Signaling 9951s, 2μM). Cells were treated with the inhibitors for 1 h at 27°C before Vip3Aa-RFP was added.

The following reagents were purchased from the indicated providers: dimethyl sulfoxide (DMSO; Sigma, D8418) and mifepristone (RU-486; Sigma, M8046). The following antibodies were used for immunoblotting: mouse anti-TurboGFP (Origene, TA150041), rabbit anti-TDP-43 (Proteintech, 10782-2-AP), rabbit anti-LC3 (MBL, PM036), mouse anti-Polyubiquitin (Enzo Life Science, BML-PW8805), mouse anti-Flag (Cell Signaling Technology, 2044), rabbit anti-HDAC6 (Santa Cruz Biotechnology, sc-11420), mouse anti-Lamin A/C (EMD Millipore, 05-714), HRP-conjugated anti-alpha-tubulin (Cell Signaling Technology, 9099), HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, sc-2004), HRP-conjugated mouse IgM (Abcam, ab97230), and HRP-conjugated mouse IgG (Santa Cruz Biotechnology, sc-2005). The following antibodies were used for immunocytochemistry (ICC): rabbit anti-cleaved caspase-3 (CC3) antibody (Cell Signaling Technology, 9664) and Alexa 594-conjugated anti-rabbit IgG (Jackson ImmunoResearch, 111-585-144). The following antibodies were used for immunohistochemistry: rat anti-ELAV (DSHB, RAT-ELAV-7), mouse anti-Polyubiquitin (Enzo Life Science, BML-PW8805), Alexa-488 conjugated rat IgG (Jackson ImmunoResearch, 112-545-167), and Alexa-594 conjugated mouse IgM (Jackson ImmunoResearch, 115-587-020).

Cell lysates or immuno-precipitated CaMKK2 were denatured in SDS sample buffer, separated by SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore). Membranes were blocked for 1 hr in PBS/1% Tween-20 (PBS-T) supplemented with 2% non-fat milk. Primary antibodies were diluted in PBS-T containing 1% non-fat milk at the following dilution: rabbit or mouse anti-Flag (Cell Signaling; Cat No 2368 S; Lot No 6; 100 ng/ml or Cat No 8146 S; Lot No 3; 100 ng/ml, respectively), rabbit anti-pThr85 (300 ng/ml)4 (link), rabbit anti-pThr172 (Cell Signaling; Cat No 2535 L; Lot No 16; 100 ng/ml), mouse anti-pan AMPK-α (Cell Signaling; Cat No 2793 S; Lot No 6; 100 ng/ml) and mouse anti-pSer antibody (BD Bioscience; Cat No 612546; Lot No 4232670; 250 ng/ml), which selectively detects phosphorylated Ser129 on CaMKK25 (link). After incubation with primary antibody solutions for 1 hr, the membranes were briefly washed in PBS-T, and then incubated with appropriate fluorescently labeled secondary antibodies, either goat anti-mouse IgG IRDye800 (Li-Cor) or goat anti-rabbit IgG IRDye680 (Li-Cor) for 1 hr. After successive washing with PBS-T, the membranes were then scanned with an Odyssey Infrared Imager (Li-Cor).

After SDS-polyacrylamide gel electrophoresis and semi-dry electro-blotting, nitrocellulose membranes (GE) were treated with 5% fat-free milk powder dissolved in PBS with 0.05% Tween 20 (PBST) as blocking agent. The primary antibodies mouse anti-Myc (#sc-40; Santa Cruz Biotechnology) or mouse anti-Flag (#8146; Cell Signaling Technology, Danvers, MA, USA) were diluted at 1:1,000 in 5% (w/v) bovine serum albumin in PBST and used for immunodetection. After incubation overnight at 4 °C, membranes were washed with PBST, exposed to the secondary horseradish peroxidase-conjugated anti-mouse antibody (#sc-2962; Santa Cruz Biotechnology; dilution 1:3,000) in 5% fat-free milk powder dissolved in PBST for 90 min at RT, washed again with PBST, and developed using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Signals were acquired using the Fusion FX7 gel documentation system (Vilber, Collégien, France). Densitometric quantification of protein bands was performed using ImageJ software (Schneider et al. 2012 (link)).

Rabbit anti-Rab5C (Proteintech, Cat. No. 27219-1-AP) and rabbit anti-TBC1D16 (Abcam, Cat. No. ab104407) were used at a dilution of 1:1000 for Western blotting. The anti-β-actin (Abcam, Cat. No. ab3280) were purchased from Abcam and used at a dilution of 1:10000 for Western blotting. Mouse anti-His (Proteintech, Cat. No. 66005-1-lg), mouse anti-Myc (Cell Signaling Technology, Cat. No. 2276S), rabbit anti-FLAG (Cell Signaling Technology, Cat. No. 14793S), mouse anti-FLAG (Cell Signaling Technology, Cat. No. 8146S) and rabbit anti-HA (Cell Signaling Technology, Cat. No. 3724S) were used at a dilution of 1:50 for immunoprecipitation. Antibody against PFV Gag was generously provided by Professor Li Zhi. The anti-Tas was produced by immunizing mice with prokaryotically expressed Tas, followed by purification according to standard procedures (18 (link)). HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit secondary antibodies were from Bioprimacy and used at 1:10,000.

Antibodies to polyclone rabbit anticaspase3, monoclonal rabbit anti-PARP1, and mouse anti-Flag were obtained from Cell Signaling Technology (USA); polyclonal mouse anti-Cul4a, monoclonal rabbit anti-α-tubulin, and anti-GAPDH were obtained from Proteintech (Wuhan, China). Protein A/G magnetic beads were acquired from Biotool (Shanghai, China).