Anti ha mouse monoclonal antibody

The trophozoites were harvested at the exponential phase of growth (5×10⁶ cells/ml) and washed twice with PBS pH 7.0. Trophozoites were re-suspended in lysis buffer (100 mM Tris–HCl pH 7.4 supplemented with 0.05 mM E64 and 1% Triton X-100) and disrupted with a hand homogenizer. Amoebic cell extracts were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond, Amersham Biosciences). Western blotting was performed using mouse anti-HA monoclonal antibody (1:500) (Invitrogen) as primary antibody and horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech) as a secondary antibody and visualized using the alkaline phosphatase conjugate substrate kit (Bio-Rad). For equal amounts of protein, the concentration was determined by the DC Protein Assay (Bio-Rad).

Cells were lysed with lysis buffer [1.85 M NaOH, 7.5% 2-Mercaptoethanol]. Proteins were precipitated with 50% Trichloroacetic acid and resuspended in Urea buffer [40 mM Tris pH8, 8.0 M Urea, 5% SDS, 1% 2-Mercaptoethanol]. Cdr1, Pdr1, and Upc2A rabbit polyclonal antibodies were previously described [18 (link),31 (link)]. Mouse anti-HA monoclonal antibody was purchased from Invitrogen. Secondary antibodies were purchased from LI-COR Biosciences. Imaging was performed with Odyssey CLx Imaging System (LI-COR Biosciences) and analyzed by Image Studio Lite Software (LI-COR Biosciences). Detected target band fluorescence intensity was normalized against tubulin fluorescence intensity and compiled from 2 biological replicate experiments and 2 technical replicates in each experiment, giving 4 replicates in total.

Protein–lipid overlay assays with PIP and lipid strips (P-6001 and P-6002 respectively, Echelon Biosciences) were performed according to the manufacturer’s instructions. Briefly, strips were first blocked with 3% fatty acid-free BSA in PBS (3 ml, 10 mM phosphate, and 150 mM NaCl, pH 7.4) for 1 h and incubated 2 h at room temperature with blocking buffer containing 0.5 μg/ml for each of GST-EXO70B1-ct, GST-EXO70B2-ct, HA-EXO70B1, and HA-EXO70B2. The strips were washed 3× with 3 ml of PBS with 0.1% Tween. To detect the proteins, an anti-GST (Echelon Biosciences, dilution 1/2000) or anti-HA mouse monoclonal antibody (Thermo Fisher Scientific, dilution 1/1,000 dilution) was used. Subsequently, chemiluminescence detection (ECL, Amersham) of the secondary anti-mouse antibody (Promega) conjugated with horse radish peroxidase was used for identification of positive interactions. The signal was documented using Bio-Rad documentation system.

Anti-FLAG mouse monoclonal antibody was purchased from Stratagene (Cat # 200472). Anti-HA mouse monoclonal antibody was purchased from Thermo Fisher Scientific (Cat #26183). Anti-GAPDH, HRP-conjugated antibody was purchased from Santa Cruz Biotechnologies (Cat #sc-365062 HRP). Goat anti-mouse and anti-rabbit HRP conjugated secondary antibodies were purchased from Azure Biosystems (Cat #AC2115) or Cell Signaling Technologies (Cat #7074S), respectively. Antibodies raised against VACV proteins A17 and A10 were gifts from Dr. Bernard Moss [65 (link)–67 (link)]. Antibodies raised against VACV protein E3 were a gift from Dr. Yan Xiang [68 (link)]. Cycloheximide(Cat#01810) was purchased from Sigma. Phenol:Chloroform:Isoamyl Alcohol (Cat#15593031) was purchased from Invitrogen. qPCR mix (Cat#QP005) was purchased from GeneCopoeia. SuperScript III Reverse Transcriptase (Cat#18-080-044), Trizol reagent (Cat#15-596-018), RNase inhibitor (Cat#AM2696), DTT (Cat#BP172) and D-Sucrose (Cat#BP220-212) were purchased from Fisher Scientific.

Primary antibodies against Actin (66,009-1-lg), HDAC4 (17449-1-AP), UB (10201-2-AP) and V5 (14440-1-AP) were purchased from Proteintech. K63-linkage-specific polyubiquitin antibody (920435621), K48-linkage-specific polyubiquitin antibody (920438081) and anti-TRIM21 antibody (92043) were ordered from Cell Signaling Technology. Anti-acetyl-Lysine antibody (06-933) was purchased from Millipore. Anti-GAC antibody (ab93434) were ordered from Abcam. Anti-HA mouse monoclonal antibody was purchased from Thermo Fisher Scientific (26183). The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene. The polyclonal antibody against anti-acetyl-lysine 311 GAC (GAC-AcK311) was manufactured by Shanghai Genomics Inc (antigen sequence: VHRYVGK(Ac)EPSGLR; immunogen: Peptide-KLH conjugated). In summary, antigen peptide (VHRYVGK(Ac)EPSGLR) was synthetized and conjugated with KLH. Then immunogen mixed with Freund's adjuvant was subcutaneously injected to rabbit every week for 4 times. Rabbit was sacrificed and serum was collected. Antibody against GAC-AcK311 was purified from serum using unacetylated antigen peptide (VHRYVGKEPSGLR).NAM (sigma, 72340) and TSA (sigma, V900931) were purchased from sigma.

Approximately 1×10⁶ transformant trophozoites were harvested 48 h after initiation of culture and washed with 2% glucose in 1X phosphate buffer saline (PBS) three times. Cells were counted and resuspended in 500 μL homogenization buffer (50mM Tris-HCl, pH 7.5, 250mM sucrose, 50mM NaCl and 0.5 mg/ml E-64 (Peptide Institute, Osaka, Japan) protease inhibitor). Cells were disrupted mechanically by a Dounce homogenizer and kept on ice for 30 min with intermittent vortexing followed by centrifugation at 500×g for 30 min at 4°C for removing the insoluble cellular debris. The supernatant, representing total cell lysate, was carefully collected. The expression of N-terminal GFP-tagged full length wild type proteins in E. histolytica trophozoites were verified by immunoblots with anti-GFP mouse monoclonal antibody (Abcam, USA), while expression of N-terminal HA-tagged full length wild type proteins were verified with anti-HA mouse monoclonal antibody (Thermo-Fisher Scientific, USA).