Rabbit polyclonal anti flag

Polyclonal rabbit anti-GFP and anti-Myc were generated as described (Mizutani et al, 2013 (link)). Mouse monoclonal glial fibrillary acidic protein (GFAP) antibody was from Chemicon (Temecula, CA, USA). Goat polyclonal anti-HSPA5, mouse monoclonal anti-GFP and anti-β-tubulin were from Santa Cruz Biotech. (Santa Cruz, CA, USA). Goat polyclonal anti-SIL1 was from abcam (Tokyo, Japan). Mouse monoclonal anti-Flag M2 and rabbit polyclonal anti-Flag were from Sigma (Tokyo, Japan). Rabbit polyclonal anti-active caspase3 and anti-Ki67 were from Cell Signaling Technology (Danvers, MA, USA) and Thermo Scientific Japan (Yokohama, Japan), respectively.

The following antisera or antibodies were used, at the indicated dilutions. Rabbit polyclonal anti-FLAG (1:2,000 Sigma Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-tobacco (Nicotiana tabacum) BIP (1:10,000 [3 (link)]); goat anti-rabbit IgG-peroxidase conjugate (1:16,000, Pierce Biotechnology Rockford, IL, USA).

Rabbit HIV-1 HXB2 integrase antisera (obtained from Dr. Duane P. Grandgenett) [30 (link)] and anti-HIV-1 integrase monoclonal antibodies were provided by the NIH AIDS Research & Reference Reagent Program (cat. #757 and #758; 8G4 cat. # 7375 and 2C11 cat. #7374). The monoclonal anti-DDX5 antibody (C10) was a generous gift from Dr. Hans Stahl (Universität des Saarlands, Sarrebruck, Germany). The following antibodies were purchased from Sigma-Aldrich (St-Louis MO): rabbit polyclonal anti-Flag, mouse monoclonal anti-Flag-M2 and mouse monoclonal β-Actin antibodies. Monoclonal anti-DDX17 antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas TX), while monoclonal anti-HDAC1 antibodies were purchased from Cell signaling Technology (Danvers MA). All secondary conjugated antibodies were provided by GE Health Care (Chicago, IL).

The proximity ligation assay (PLA) (Söderberg et al, 2006 (link)) was performed using the Duolink^® PLA kit (Sigma Aldrich), according to the manufacturer’s protocol. Briefly, HEK293 cells expressing FLAG- and HA-tagged proteins were fixed for 10 min in 4% paraformaldehyde, permeabilized for 20 min in PBS containing 0.5% Triton X-100 and 3% BSA, and incubated for 18–24 h at 4˚C with the anti-FLAG rabbit polyclonal (Sigma-Aldrich) and anti-HA mouse monoclonal (Covance) antibodies. Cells were then incubated for 1 h at 37 ˚C in a humidified chamber with the PLA probes: Duolink^® In Situ PLA^® Probe Anti-Mouse MINUS (Sigma Aldrich) and Duolink^® In Situ PLA^® Probe Anti-Rabbit PLUS (Sigma Aldrich). Using Duolink^® In Situ Detection Reagents Orange (Sigma Aldrich), ligation reaction was performed for 30 min at 37 ˚C, followed by amplification for 100 min at 37 ˚C. For visualization of the primary antibodies, cells were incubated for 45 min at 25 ˚C with Alexa Fluor 488-labeled goat anti-rabbit (Thermo Fisher Scientific) and Alexa Fluor 647-labeled goat anti-mouse IgG antibodies (Thermo Fisher Scientific).