Mouse monoclonal anti flag m2 antibody

HEK 293T cells were transfected at 60–80% confluence in Nunc Lab-Tek II Chamber Slides (ThermoFisher Scientific) using the jetOPTIMUS transfection kit (Polyplus Transfection) with either pCDNA3.1-V5-dSpCas9 or pCDNA3.1-3xFLAG-dSpCas9. Slides were fixed with MeOH, blocked for 1 h at room temperature, and incubated under gentle orbital shaking with primary antibody overnight: either V5 Tag mouse monoclonal antibody (ThermoFisher Scientific #R960-25) at 1:3000 dilution or mouse monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich #F1804) at 1:1000 dilution. Slides were washed five times for 10 min with phosphate-buffered saline with Tween 20 (PBST), then incubated for 1 h at room temperature under gentle orbital shaking with secondary antibody: Goat anti-mouse IgG AlexaFluor 488 Superclonal Recombinant Secondary antibody (ThermoFisher Scientific #A28175) at 1:2000 dilution. Slides were washed five times for 10 min with PBST, then washed three more times with PBS before mounting overnight with 4',6-diamidino-2-phenylindole (DAPI). All antibodies were incubated with 5% BSA in 0.1% Tween-PBS. Immunofluorescence images were taken at 63x objective with a Zeiss LSM 780 confocal microscope in 5–10 slices, with maximum intensity projections across the entire image plane generated in Zeiss ZEN 2010 for figures.

Yeast cells expressing FLAG-tagged protein were used. The protein extraction was described elsewhere⁶¹ . Proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Protran) (GE healthcare, Little Chalfont, Buckinghamshire, England). The protein level in each SDS-PAGE lane was normalized and then confirmed using 0.1% Ponceau S solution (Sigma-Aldrich, St. Louis, MO, USA). Mouse monoclonal anti-FLAG M2 antibody (1:1000 dilution) (Sigma-Aldrich, St. Louis, MO, USA) was used to detect FLAG-tagged protein.

Broad-spectrum cysteine protease inhibitor E64D (#HY-100229), Cathepsin L-specific inhibitor SID 26681509 (#HY-103353), Cathepsin B-specific inhibitor CA-074 (#HY-103350), PIKfyve inhibitors apilimod (#HY-14644) and YM201636 (#HY-13228), were purchased from Med Chem Express (MCE, New Jersey, USA). Calcium channel blocker tetrandrine (#S2403) was purchased from Selleck Chemicals (Texas, USA). Endosome acidification inhibitors NH₄Cl (#A9434) and bafilomycin A (#19-148) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China). Goat polyclonal against human ACE2 antibody (#AF933) was purchased from R&D Systems (Minnesota, USA). Mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich. Donkey Anti-Rabbit IgG (#711-035-152), Goat Anti-Mouse IgG (#115-035-146), and Rabbit Anti-Goat IgG (#305-035-003) were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG were purchased from ZSGB-Bio LLC (Beijing, China).

We prepared constructs for co-immunoprecipitation and immunofluorescence experiments. Mouse entire coding regions of wild-type Slc35d3 (RefSeq, NM_029529, http://www.ncbi.nlm.nih.gov/refseq/), D1r (RefSeq, NM_010076), D2r (RefSeq, NM_010077), and human SLC35D3 (RefSeq, NM_001008783) were subcloned into the pEGFP-C2 or -N2 vector (with GFP-tag), pCMV-tag2B vector (with Flag-tag) and pCMV-tag3B vector (with Myc-tag) as specified in the figures. The fragments of mouse SLC35D3 and mouse D1R specified in figure legends were generated by subcloning. The mutant human SLC35D3 constructs (ΔK404 and insL201) were generated by site-directed mutagenesis (Takara, Japan) using human wild-type SLC35D3 construct.
Transfected HEK-293T cells grew to confluency on 6-well plates. Cells were harvested and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitors. Cell lysates were centrifuged at 18,000 g for 10 min, and the supernatant was collected and recentrifuged. The supernatant was incubated overnight with 3 µg mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) and washed 6 times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl). The samples were eluted with elution buffer (5 µg/µl 3× FLAG peptide) and subjected to SDS-PAGE and Western blotting with anti-Myc or anti-Flag antibody as described above.