Hrp conjugated anti ha antibody

10.10⁶ cells were harvested, washed with PBS, and resuspended in lysis buffer (400 mM NaCl, 10 mM HEPES, pH 7.5, 0.1% NP‐40, Sigma P8340); the NaCl concentration was then brought down to 150 mM; and samples were sonicated using a probe sonicator and then centrifuged at 17,000 rcf in order to remove insoluble material. The protein concentration of the lysates was measured using a BCA assay (Thermo Fisher, 23225), and equivalent protein quantities were then incubated with 50 μl of pre‐washed anti‐HA agarose beads (Sigma, A2095) overnight on a rotating wheel at 4°C. The samples were then incubated five times for 10 min with 1 ml of wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 0.1% NP‐40, protease inhibitors) on a rotating wheel at 4°C. Immunoprecipitates were eluted in Laemmli buffer at 95°C for 10 min. Similar elution volumes and protein quantities, for the IPs and the inputs, respectively, were loaded on a gel. Western blots were performed using anti‐Sirt1 (Abcam, ab32441), anti‐IPO7 (Abcam, ab99273), anti‐GFP (Abcam, ab5450), anti‐KAP1 (Merck, MAB3662), HRP‐conjugated anti‐Flag antibody (Sigma, A8592), HRP‐conjugated anti‐HA antibody (Sigma, 12013819001), HRP‐conjugated anti‐goat antibody (Dakocytomation, P0449), HRP‐conjugated anti‐rabbit antibody (Santa Cruz, sc‐2004), and HRP‐conjugated anti‐mouse antibody (GE Healthcare, NA931V).

First, 293T cells were cotransfected with plasmids encoding FLAG- or hemagglutinin (HA)-tagged TSG101 and RABV M using polyethylenimine. Cell lysates were harvested at 72 h posttransfection and clarified via centrifugation. Immunoprecipitation was then conducted using anti-HA magnetic beads (Pierce) according to the manufacturer’s protocol. The precipitates were extracted into 1× SDS sample buffer containing 2-mercaptethanol and subjected to a standard immunoblotting assay using horseradish peroxidase (HRP)-conjugated anti-HA antibody (catalog no. H6533; Sigma; 1:3,000 dilution) and anti-FLAG antibody (catalog no. A8592; Sigma; 1:3,000 dilution).

Cas2-Flag on pET-28a, FtsZ-HA on pET-11a and the vector controls were co-transformed into E. coli BL21-AI.^{16 (link)} Stable transformants selected on LB plates with kanamycin (50 μg/ml) and ampicillin (100 μg/ml) were cultured in LB medium with the two antibiotics plus 0.2% (wt/vol) L-arabinose and 0.2 mM IPTG at 37 °C for 3 h. Cells were pelleted by centrifugation at 4000 rpm for 5 min and subjected to a standard ultra-sonication protocol described in an early publication.^{26 (link)} The supernatants were collected after a 10 min centrifugation at 12,000 rpm, and the same protocol was repeated 2 times. The lysates without debris were subjected to an immunoprecipitation assay^{23 (link)} with anti-HA beads (Millipore catalogue number IP 0010) for FtsZ and anti-Flag beads (Millipore catalogue number FLAGIPT1) for Cas2. The total proteins eluted from the beads were separated on 4–12% Bis-Tris Plus gels (Invitrogen catalogue number NW04122BOX) with MES SDS running buffer (Invitrogen catalogue number NP0002); transferred onto a PVDF membrane (Invitrogen catalogue number IB24001) by iBlot II (Invitrogen); and subjected to standard western analysis with an HRP-conjugated anti-Flag antibody (Sigma-Aldrich catalogue number A8592-1MG) and HRP-conjugated anti-HA antibody (Sigma-Aldrich catalogue number H6533).