Anti ha monoclonal antibody

Cdc34 fusion proteins were expressed under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase, GPD, promoter from a CEN plasmid (p416 GPD) with a URA3 selection marker in S. cerevisiae strain BY4741 pdr5Δ (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pdr5::kanMX4). Cells were grown to mid-log phase and lysed by vortexing with glass beads (BioSpec Products). Protein extracts were prepared and analyzed by Western blotting using standard protocols as described^{6 (link)}. Cdc34 protein was detected with a monoclonal anti-HA antibody (1:5,000; Sigma, #H9658) and an Alexa-800-labelled goat anti-mouse secondary antibody (1:20,000; Rockland Immunochemicals, #610-132-121), and Scs2p was detected by an anti-SCS2 polyclonal antibody (1:1,000; gift from J. Brickner, Northwestern University) and an Alexa-680 goat anti-rabbit secondary antibody (1:20,000; Invitrogen, #A21109). Protein amounts were estimated by direct infrared fluorescence imaging (Odyssey LICOR Biosciences). Original images of Western blots can be found in Supplementary dataset 1.

DF1 cells were co-transfected with pRK5-HA-Ub, pRK5-HA-Ub-K48 or pRK5–HA–Ub-K63 plasmids and the indicated amounts of IBV PLP, PLP-TM and specific catalytic mutants. The cells were then lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, China). The cell lysates were analyzed for HA-conjugated proteins using western blotting with monoclonal anti-HA antibody (1:5000, Sigma). An anti-N antibody was used to detect the viral replication level. To confirm the expression levels of PLP and the mutants, anti-flag antibody (1:5000, Sigma) was used to detect the flag-tagged proteins. Actin was detected using β-actin antibody (1:5000, Sigma) as a protein loading control.

Cell surface ELISAs were performed as previously described ^{67 (link)}. HEK cells were treated with the Gα_s siRNA duplex and transfected with HA-CXCR4 or HA-DOP (as described above, 2 μg/P10 dish). Forty-eight hours after the initial siRNA treatment, the cells were plated onto poly-L-Lysine-coated 24-well plates (Sigma-Aldrich, Saint Louis, MO, USA). After 24 h, the cells were starved for 1 h at 37°C in DMEM and then incubated with DMEM containing 25 mM Hepes, 0.2% BSA and agonist (100 nM SDF1-α or 5 μM DPDPE) for 30 min or agonist for 30 min followed by antagonist (10 μM Naloxone or AMD3100) for 60 min. The cells were then fixed with 3% formaldehyde, washed with TBS, blocked in 5% BSA and incubated for 1 h with primary antibody (monoclonal anti-HA antibody) and for 45 min with secondary antibody (alkaline phosphatase-conjugated goat anti-mouse antibody; Sigma-Aldrich, Saint-Louis, MO, USA). The cells were then washed three times, and 250 μl of a colorimetric alkaline phosphatase substrate (diethanolamine and phosphatase substrate; Sigma-Aldrich, Saint-Louis, MO, USA) was added. The plates were incubated at 37°C for the appropriate time, and then 250 μl of NaOH (0.4 M) was added to stop the reaction. A 100-μl aliquot of the colorimetric reaction was collected, and the absorbance was measured at 405 nm using a spectrophotometer (Titertek Multiskan MCC/340; Labsystems).