The Myc-Cdc5L and EGFP-securin plasmids were constructed for immunoprecipitation (IP). According to the previous description, the plasmids were transfected into 293T (Li et al., 2019 (link)). After 48 h, we collected the cells with 400-μl IP lysis buffer (Thermo Fisher Scientific, 88804) with the cocktail of protease and phosphatase inhibitors, followed by incubation on ice for 20 min. Next, we centrifuged the lysates at 13,400 rpm for 10 min at 4°C, and 400 μl of the supernatant was incubated with 1-μg primary antibody (GFP: abcam, ab290 or Myc: sigma, M4439) and 20-μl Magnetic Beads (Thermo Fisher Scientific, 88804) at 4°C overnight with rotation. The next morning, after washing five times, 40-μl 1 × sodium dodecyl sulfate loading buffer was added to the immunoprecipitate, and Western blotting was prepared. Western blot experiment was performed as described previously (Zhang et al., 2021 (link)). The anti-β-actin antibody (ZSGB-BIO, TA-09,1:1,000), rabbit anti-Cdc5L antibody (Thermo, 702831, 1:500), mouse monoclonal anti-Myc antibody (Sigma, M4439, 1:3,000), mouse monoclonal anti-Cyclin B1 antibody (Abcam, ab72, 1:1,000), and anti-SMC3 antibody (Abcam, ab128919, 1:1,000) were used for Western blotting.
M4439
Manufactured by Merck Group
Sourced in United States, United Kingdom, Switzerland
M4439 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose instrument for scientific applications. The core function of this product is to facilitate various laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
F3165, by Merck Group (5 mentions)
F1804, by Merck Group (5 mentions)
A11001, by Thermo Fisher Scientific (3 mentions)
H3663, by Merck Group (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti myc, by Merck Group (3 mentions)
A11122, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
H6908, by Merck Group (2 mentions)
H6533, by Merck Group (2 mentions)
A4596, by Merck Group (2 mentions)
A8592, by Merck Group (2 mentions)
A21203, by Merck Group (2 mentions)
B2261, by Merck Group (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab2365, by Abcam (1 mentions)
Ab140290, by Abcam (1 mentions)
Ab58310, by Abcam (1 mentions)
48 protocols using m4439
1
Immunoprecipitation of Myc-Cdc5L and EGFP-securin
2
Cell Culture and Antibody Reagents
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HEK293T cells, L cells (an immortalized mouse fibroblast cell line), SW480 cells and HCT116 cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China). All cells were grown in DMEM medium (Invitrogen, Carlsbad, CA), and maintained in culture supplemented with 10% heat-inactivated fetal calf serum, 100 ug ml−1 of penicillin, and 100 μg ml−1 of streptomycin at 37 °C with 5% CO2 in a humidified incubator (Thermo, Waltham, MA). During the study, all cell cultures were periodically tested for mycoplasma by using MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME). For western blot, antibodies specific for VGLL4 (1:500, ab140290), TEAD4 (1:500, ab58310), Histone H3 (1:1,000, ab6002) and β-catenin (1:1,000, ab2365) were purchased from Abcam (Cambridge, UK); those for FLAG (1:5,000, M4439) and α-tubulin (1:2,000, T6199) were from Sigma (St. Louis, MO); and those for TCF4 (1:500, sc-166699), c-Jun (1:1,000, sc-4113) and GST (1:1,000, sc-138) were bought from Santa Cruz Biotechnology (Santa Cruz, CA).
3
Opioid Receptor Colocalization Assay
4
Immunofluorescence Staining of Myc and GFP
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Cells were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated overnight at 4°C with primary antibodies anti‐Myc (1:500, M4439, Sigma) o anti‐GFP (1:750, A11122, Life technologies). For anti‐myc, binding was visualized using Alexa 488 secondary antibody; anti‐GFP binding was visualized using biotinylated secondary antibody/Cy3‐conjugated streptavidin.
5
Recombinant Erc1 Protein Expression in Pichia
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The Pichia pastoris KM71H-OCH protein expression system was used to produce N-terminally His and C-terminally Myc-His tagged U. maydis Erc1, Erc1M1x, and Erc1M2x recombinant proteins. All genes for each respective protein were cloned into the pGAPZαA vector (Invitrogen; Carlsbad, USA) under the control of a constitutive promotor with an α-factor signal peptide for secretion. Protein expression was performed by growing Pichia in 1 L buffered (100 mM sodium phosphate buffer, pH 6.0) YPD medium at 28 °C for 48 hours with 200 rpm shaking (pGAPZαA, B, & C Pichia pastoris Expression Vectors, Invitrogen; Carlsbad, USA). Recombinant protein purification was performed with a Ni-NTA-matrix (Ni-Sepharose™ 6 Fast-Flow, GE-Healthcare; Freiburg, Germany). After protein purification, each protein sample was applied to the NAP-25 column to exchange buffer with 20 mM potassium phosphate buffer pH 6.0. The proteins were stored at −20 °C for further experiments. Western blot analysis was performed by using anti-His (H1029, Sigma-Aldrich, Steinheim) and anti-Myc (M4439, Sigma-Aldrich, Steinheim) antibodies with 1:5000 and 1:3000 dilution, respectively20 (link). As a secondary antibody, anti-mouse IgG HRP (#7076, Cell Signaling Technology) was used in 1:3000 dilution20 (link).
6
Visualizing MEF2C and UBE3A Localization
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Expression plasmids expressing human MEF2C and UBE3A and respective negative control plasmids were obtained from Sino Biologicals (MEF2C-HA: HG12320-CY, UBE3A-Myc:HG11130-CM, pCMV-Myc:CV014 and pCMV-HA:CV013) and used for transient transfection. Transfected HeLa cells were grown on poly-lysine coated coverslips, fixated with 4% paraformaldehyde in PBS for 10 minutes and stained with anti-Myc (M4439, Sigma-Aldrich) and anti-HA (H6908, Sigma-Aldrich) and with Alexa Fluor™ 488 goat anti–mouse and Alexa Fluor™ 488 donkey anti–rabbit antibodies (A11001 and A10040, Thermo Fisher). Nuclei were counterstained with DAPI (Serva). Images were taken with a Zeiss Axio Imager Z2 Apotome microscope with a 63x objective and analyzed in ImageJ.
7
Antibody Production and Characterization
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Antibodies to c-myc- (M4439), HA- (H3668), and FLAG (M5, F4042)-tags were purchased from Sigma. Rabbit antibodies against HEV-LP10 (link) and peptides corresponding to Japanese encephalitis virus (JEV) E protein epitopes, amino acid residues from 337 to 345 (JEV1 epitope) and amino acids from 362 to 369 (JEV2 epitope) were prepared by Scrum Inc. (Tokyo, Japan).
8
Immunoblotting Analysis of Plant Proteins
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Total proteins were extracted from rosettes homogenized in 50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM DTT, 1× Complete Protease Inhibitor EDTA-free Mixture (Roche). Equal amounts of soluble proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and subject to immunoblotting. For stable lines expressing YFP-tagged proteins, anti-GFP/YFP (rabbit, ab290; Abcam, Cambridge, UK) and anti-plant actin (mouse, A0480; Sigma-Aldrich) were used. For immunoblotting of BAM9, we used antibodies raised in rabbits (Eurogentec, Seraing, Belgium) against the recombinant purified protein. For immunoblotting of BAM3, we used antibodies described previously (Fulton et al., 2008) (link). Proteins were detected using near-infrared fluorescence from IRDye secondary antibodies (anti-rabbit and/or anti-mouse) and an Odyssey CLx detection system (Li-Cor, Lincoln, NE, USA). For the stable BAM9-TAP lines, an anti-myc (mouse, M4439; Sigma-Aldrich) antibody was used to detect the proteins followed by colorimetric detection.
9
EST12 Protein Treatment in RAW264.7 Cells
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RAW264.7 cells were pretreated with MG-132 (5 μM) for 1 h and treated with 2 μM purified EST12 protein for indicated time. Media was removed from stimulated macrophages and the dishes were submerged in 4% paraformaldehyde (PFA). The fixed dishes were submerged in PBS to remove residual PFA and then cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature (RT), washed in PBS and blocked in PBS containing 10% donkey serum. Primary antibodies against FBW7 (28424-1-AP, Proteintech, China) and Myc (M4439, Sigma-Aldrich, USA) were used at a 1:50 dilution in PBS with 3% serum and incubated at 4°C overnight. Then Alexa Fluor 594-conjugated goat anti-rabbit IgG (A-11037, ThermoFisher, USA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (A28175, ThermoFisher, USA) was used at 1:500 dilution in PBS with 3% FBS and incubated at RT for 1 h. Dishes were washed to remove the unbound secondary antibody and the cellular nuclei DNA was stained with DAPI. Fluormount-G Anti-Fade Mounting Medium (Southern Biotech) was added to each well to protect the fluorescent signal. Confocal fluorescence microscopy was performed using ZEISS LSM880 microscope and representative images were captured.
10
Probing SEAP1 protein interactions
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