Truncated mutants of GP73 were designed, as shown in Fig. 6a . Mutants were constructed based on the pCMV-c-FLAG plasmid. Protein extraction, immunoprecipitation and immunoblotting were performed, as described above. Truncated mutants of GP73 were immunoprecipitated using rabbit anti-FLAG pAb and detected using mouse anti-FLAG mAb (Sigma-Aldrich Co., St. Louis, MO, USA).
Mouse anti flag mab
Manufactured by Merck Group
Sourced in United States, China
The Mouse anti-FLAG mAb is a monoclonal antibody that recognizes the FLAG epitope tag. It is commonly used in various laboratory applications, such as immunoprecipitation, Western blotting, and immunohistochemistry, to detect and purify proteins tagged with the FLAG sequence.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin mab, by Merck Group (5 mentions)
Mouse anti ha mab, by Merck Group (5 mentions)
Rabbit anti flag pab, by Merck Group (3 mentions)
Rabbit anti myc pab, by Merck Group (3 mentions)
Mouse anti myc mab, by Merck Group (3 mentions)
Mouse anti ha mab, by Abmart (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti tubulin mouse mab, by Fortrea (3 mentions)
Mouse anti α tubulin mab, by Merck Group (2 mentions)
Mouse anti α tubulin, by Abcam (2 mentions)
Mouse anti gfp mouse mab, by Affinity Biosciences (2 mentions)
Mouse anti laminb1 mab, by Proteintech (2 mentions)
Goat anti mouse ird800 conjugated mab, by Merck Group (2 mentions)
Criterion tgx precast gel, by Bio-Rad (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ha mab, by Cell Signaling Technology (2 mentions)
Irdye conjugated secondary antibody, by LI COR (2 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gapdh mabs, by Abcam (2 mentions)
Mouse igg binding protein conjugated to hrp mabs, by Abmart (2 mentions)
36 protocols using mouse anti flag mab
1
Truncated GP73 Mutants Analysis
2
Culturing HEK293T cells and PAMs for ASFV research
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HEK293T cells and PAMs, which were stored in our laboratory, were cultured in RPMI 1640 medium (catalog no. C11875500BT; Gibco) supplemented with 5% antibiotics-antimycotics (10,000 IU/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B) (catalog no. 15240-062; Gibco) and 10% heat-inactivated fetal bovine serum (FBS) (catalog no. 10099-141C; Gibco) in a 37°C incubator in 5% CO2. The ASFV pig/Heilongjiang/2018 (ASFV HLJ/2018) strain was isolated from field samples from China as described previously (63 (link)). ASFV-ΔH240R was generated in our previous study (8 (link)).
A rabbit anti-Myc PAb (catalog no. ab9106; Sigma-Aldrich), a mouse anti-Flag MAb (catalog no. ab62928; Sigma-Aldrich), and a rabbit anti-Flag PAb (catalog no. ab1162; Sigma-Aldrich) were purchased from Sigma-Aldrich. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (catalog no. 2072687; Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 1942237; Invitrogen), Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 111-585-003; Invitrogen) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (catalog no. 175697; Invitrogen) antibodies were purchased from Invitrogen. A mouse anti-GST MAb (catalog no. K200006M; Solarbio) and 4′,6-diamidino-2-phenylindole (DAPI) (catalog no. C006; Solarbio) were purchased from Solarbio.
A rabbit anti-Myc PAb (catalog no. ab9106; Sigma-Aldrich), a mouse anti-Flag MAb (catalog no. ab62928; Sigma-Aldrich), and a rabbit anti-Flag PAb (catalog no. ab1162; Sigma-Aldrich) were purchased from Sigma-Aldrich. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (catalog no. 2072687; Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 1942237; Invitrogen), Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 111-585-003; Invitrogen) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (catalog no. 175697; Invitrogen) antibodies were purchased from Invitrogen. A mouse anti-GST MAb (catalog no. K200006M; Solarbio) and 4′,6-diamidino-2-phenylindole (DAPI) (catalog no. C006; Solarbio) were purchased from Solarbio.
3
Antibody and Inhibitor Use in Western Blot
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The following antibodies were used in this study: mouse anti-Flag mAb (used 1:1000 in WB, F1804), rabbit anti-GAPDH mAb (used 1:10000 in WB) (G9545), mouse anti-α-tubulin mAb (used 1:10000 in WB, 05–829) and mouse anti-vinculin (used 1:500 in WB, V9131) from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-DLC1 mAb (used 1:500 in WB and IF, 612021) and mouse anti-FAK mAb (used 1:1000 in WB, 610088) from BD Biosciences (Heidelberg, Germany); rabbit anti-paxillin pAb (used 1:500 in WB and IF, sc-5574) and mouse anti-His mAb (used 1:200 in WB, sc-8036) from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-USP7 mAb (WB 1:1000, 4833), rabbit anti-GFP mAb (WB 1:1000, 2956) and rabbit anti-HA mAb (WB 1:1000, 3724) were from Cell Signaling (Danvers, MA, USA); rabbit anti-HECTD1 pAb (WB 1:1000, 20605–1-AP) was from Proteintech (Manchester, UK). HRP-labeled secondary anti-mouse and anti-rabbit IgG antibodies were purchased from and Dianova (Hamburg, Germany), Alexa-Fluor®-labeled secondary IgG antibodies were from Invitrogen (Carlsbad, CA, United States). Inhibitors used were: Bortezomib from UBPBio (Dallas, TX), MG-132 from Selleck Chemicals (Houston, TX); PR-619, HBX 41108 and P5091 from Cayman Chemicals (Ann Arbor, MI); cycloheximide from Santa Cruz (Dallas, TX).
4
Immunofluorescence Assay for E2 and MEK2 Proteins
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HEK293T cells were transiently cotransfected with pCAGGS-E2-Flag (1 μg) and pMyc-MEK2 (1 μg). PK-15 cells were mock infected or infected with CSFV at an MOI of 0.1. At 36 hpt or 48 hpi, the plasmid-transfected or CSFV-infected cells were fixed with 4% paraformaldehyde and permeabilized with 0.15% Triton X-100. The cells were further incubated with a mouse anti-Flag MAb (catalog no. F1804; Sigma-Aldrich) or anti-E2 MAb HQ06 (31 (link)) for 1.5 h, followed by incubation with a rabbit anti-Myc PAb (catalog no. C3956; Sigma-Aldrich) or a rabbit anti-MEK2 PAb (catalog no. sc-525; Santa Cruz). Subsequently, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (catalog no. F2012; Sigma-Aldrich) and tetramethyl rhodamine isocyanate-conjugated goat anti-rabbit IgG antibody (whole molecule) (catalog no. T6778; Sigma-Aldrich). After incubation with 4,6-diamidino-2-phenylindole (DAPI), the cells were observed using a Leica SP2 confocal system (Leica Microsystems; Germany).
5
Amplification and Cloning of STAT3 and BLCAP
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Human full-length STAT3 cDNA was amplified from a whole human cDNA library and was cloned into PCAGGS-HA with specific primers (Supplementary Table 1 ), The recombinant pEF-BLCAP-3×FLAG plasmid was established and stored in our laboratory. Two edited versions of BLCAP were amplified by PCR with site specific primers (Supplementary Table 1 ) from the plasmid pEF-BLCAP-3×FLAG. All constructs were confirmed by DNA sequencing. The antibodies used in this study included: mouse anti-FLAG mAb, rabbit anti-HA mAb (both from Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-human mAbs against STAT3, p-STAT3 (both from Cell Signaling Technology, Danvers, MA, USA); mouse anti-β-actin, rabbit anti-human mAbs against STAT1, p-STAT1, Bcl-2, Mcl-1 and survivin (both from Ruiying Biological, Suzhou, China). Mouse control IgGs was purchased from Santa Cruz Biotechnology (CA, USA). Human IL-6 was purchased from Proteintech (Proteintech Group, Wuhan, China).
6
Antibody Characterization for Cell Biology
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A rabbit anti–l-afadin pAb was prepared as described previously (Mandai et al., 1997 (link)). The Abs listed below were purchased from commercial sources: rabbit anti–α-catenin pAb (C2081; Sigma-Aldrich); mouse anti–α-actinin mAb (A5044; Sigma-Aldrich); rabbit anti–β-catenin pAb (C2206; Sigma-Aldrich); mouse anti-FLAG mAb (F3165; Sigma-Aldrich); rabbit anti-GAPDH mAb (14C10; Cell Signaling Technology); rabbit anti-myosin light chain (phospho S20) pAb (ab2480; Abcam); rabbit anti-sodium potassium ATPase pAb (ab76020; Abcam); rabbit anti-nonmuscle myosin heavy chain II-B pAb (PRB-445P; BioLegend); mouse anti-vinculin mAb (V9264; Sigma-Aldrich); and rat anti–ZO-1 mAb (sc-33725; Santa Cruz Biotechnology). A rat anti–E-cadherin mAb (ECCD2) was a kind gift from M. Takeichi. The HRP-conjugated secondary Abs used for Western blotting were purchased from GE Healthcare. The Alexa Fluor–conjugated secondary Abs used for immunocytochemistry were purchased from Thermo Fisher Scientific.
7
EIAV Protein Detection in eMDM Cells
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Mat proteins in eMDM cells infected with EIAVDLV121 and EIAVDLV34 were detected using a mouse anti-Mat monoclonal antibody (1G3), which was produced by immunization of mice with a Keyhole Limpet Hemocyanin (KLH) carrier protein containing a polypeptide corresponding to the C-terminal 15 amino-acids (ENAKSSYISCNNASI) of Mat. Gag proteins were analyzed using a mouse anti-p26 MAb (9H8), kept in our lab (61 (link)). The primary antibodies purchased from commercial sources are listed below: mouse anti-HA MAb (Sigma, USA), mouse anti-FLAG MAb (Sigma, USA), mouse anti-GFP mouse MAb (Affinity Bioscience, USA), mouse anti-LaminB1 MAb (Proteintech, China), mouse anti-α tubulin (Abcam, UK), and mouse anti-β actin MAb (Sigma, USA). Goat anti-mouse IRD800-conjugated MAb (Sigma, USA) was used as a secondary antibody for Western blotting.
8
PRRSV Protein Interaction Analysis
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HDAC2 was amplified from PAMs’ cDNA using the primers listed in Table 1 and subcloned into the pCAGGS vector with a C-terminal HA tag. The recombinant pCAGGS plasmids encoding individual PRRSV viral proteins (nsp1α, nsp1β, nsp4, nsp5, nsp7, nsp9-11, ORF2α, ORF5, and ORF7) were fused with a Flag tag and were already available in our laboratory. Site-directed mutagenesis was employed to generate mutant versions of the PRRSV nsp11 construct, specifically the C112A, H129A, H144A, and K173A mutations. These mutant plasmids were created using a mutagenesis kit (TakaRa, Dalian, China). All plasmids were then confirmed by Sanger sequencing to ensure accuracy. For this study, we utilized a range of commercial antibodies. The HDAC2 mouse monoclonal antibody (mAb) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-Flag mAb, Mouse anti-HA mAb, and Mouse anti-β-actin mAb were purchased from Sigma-Aldrich (Sigma, Northbrook, IL, USA). IRDye-conjugated secondary antibodies were sourced from Li-Cor Biosciences. Additionally, a mouse anti-PRRSV N protein mAb was generated and purified specifically for our laboratory’s use.
9
ATM Kinase Activity Assay in Irradiated Cells
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ATM-deficient GM16666 cells in log growth phase were plated on a 10-cm plate at 80% confluency and calcium-phosphate co-transfected with 5 μg of pcDNA3.1(+)Flag-His-ATM wt or pcDNA3.1(+)Flag-His-ATM kd plasmid DNA per plate and 100 ng of psi-miR-34 WT or psi-miR-34 MT43 (link). Following a 24-h incubation, the cells were exposed to 2 Gy of ionizing radiation. Cells were harvested at 6 h post IR. Plates were washed with PBS and the cells were lysed in wells with Passive Lysis Buffer for 30 min on ice. Lysates were centrifuged at 16,000 g at 4 °C, for 15 min and the supernatant was transferred to a fresh tube. 20 μl of lysate was reserved for luciferase reporter activity (as described above); 80 μl of lysate was boiled in SDS Sample buffer and samples were separated using a 4–20% Criterion TGX Precast Gel (Bio-Rad). Protein was transferred to Whatman BA85 Nitrocellulose and protein was detected using: mouse anti-FLAG mAb (F3165, Sigma, 1:10,000) and sheep anti-mouse IgG, HRP-linked Ab (NA931, GE Healthcare, 1:50,000). Pierce ECL western blotting substrate (PI-32109) was used for detection (shown in Supplementary Fig. 11 ).
10
Antibody Usage in Immunodetection Experiments
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The following antibodies were used in this study. Rabbit anti-HA monoclonal antibody (MAb) (#3724), rabbit anti-Myc MAb (#2278), anti-mouse IgG HRP-linked antibody and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA). Mouse anti-FLAG MAb (#F1804) was obtained from Sigma-Aldrich (MO, USA). Rabbit anti-GAPDH polyclonal antibody (PAb) (#10494-1-AP) and rabbit anti-cGAS PAb (#26416-1-AP) were purchased from Proteintech Group (Chicago, IL). ALexa Flour 633 goat anti-mouse IgG(H+L) (#A21052) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse anti-SVV 3C polyclonal Ab was prepared by our laboratory.
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