For the in vitro kinase assays, 2 μg of dephosphorylated BCAP (FL) or 100 μg of dephosphorylated myelin basic protein (31314; Active Motif) were diluted in 500 μl of kinase buffer (50 mm HEPES, 10 mm MgCl2, 0.01% BRIJ35, 1 mm EGTA, and 150 μm ATP, pH 7.5). Upon adding 60 pmol of SYK (PV3857; Thermo Fisher Scientific), LYN (PV6448; Thermo Fisher Scientific), BTK (PV3363; Thermo Fisher Scientific), TYK2 (PV4790; Thermo Fisher Scientific), ITK (PV4193; Thermo Fisher Scientific), CSNK1A1 (PV3850; Thermo Fisher Scientific), or CSNK2A1 (PV3248; Thermo Fisher Scientific), the samples were incubated at 30 °C for 30 min. The reaction was stopped using 4× SDS loading dye, and the samples were analyzed using Western blotting. For chemiluminescence detection, anti-BCAP (AF4857; R&D Systems), anti-phosphotyrosine (Ab179530; Abcam), anti-phosphoserine (Ab9332, Abcam), anti-rabbit IgG-HRP (A0545; Sigma), and anti-goat IgG-HRP (A5720; Sigma) were used.
Ab179530
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab179530 is a monoclonal antibody that recognizes the Cytokeratin 18 protein. Cytokeratin 18 is an intermediate filament protein expressed in simple and stratified epithelial cells. The antibody can be used for the detection of Cytokeratin 18 in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab9332, by Abcam (3 mentions)
Ab108414, by Abcam (2 mentions)
Csnk1a1, by Thermo Fisher Scientific (1 mentions)
Af4857, by R&D Systems (1 mentions)
Anti rabbit igg hrp, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mg132, by MedChemExpress (1 mentions)
Nitrocellulose membrane, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab51608, by Abcam (1 mentions)
Hrp conjugated goat anti mouse igg ab97040, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Sample buffer, by Merck Group (1 mentions)
Coomassie blue, by Bio-Rad (1 mentions)
Ab218195, by Abcam (1 mentions)
Ufc901096, by Merck Group (1 mentions)
Ab126704, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
6 protocols using ab179530
1
In Vitro Kinase Assay for BCAP Phosphorylation
2
Tie2 Phosphorylation Assay in 293T Cells
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Wild-type (WT) or mutant TEK-Flag plasmids (1 μg) were transfected into subconfluent 293T cells in six-well dishes using Lipofectamine 2000 (Life Technologies, Carlsbad, CA). Twenty-four hours later, cells were lysed by 1% Triton X-100, centrifuged, and separated into supernatant and insoluble pellets. Radioimmunoprecipitation assay (RIPA) buffer was added to the pellets before sonication. Same amount of proteins (15 μg) was separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, antibodies against phosphotyrosine (1:3,000; ab179530, Abcam, Cambridge, United Kingdom) and Flag (1:5,000; 20543-1-AP, Proteintech, Rosemont, IL, United States) were used to detect the phosphorylation and expression of Flag-tagged Tie2, respectively. β-Actin (1:5,000; 66009-1-Ig, Proteintech, Rosemont, IL, United States) was used as a loading control. For proteasomal inhibition assay, transfected 293T cells were treated with 5 μM MG132 (MedChemExpress, Shanghai, China) for 24 h before harvest.
3
Western Blot Analysis of Cell Proteins
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Cell lysate proteins were resolved by SDS-PAGE using precast 4%–20% NuPAGE Bis–Tris Gels (Thermo Fisher Scientific, Waltham, MA) and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) using the standard immunoblotting technique. nitrocellulose membranes were processed with different specific primary antibodies, e.g., anti-HIF-1α (ab51608), anti-β actin (ab8227), anti-band 3 (ab108414), anti-phosphotyrosine (ab179530), and anti-phospho (Y359)–band 3 (ab77236) (Abcam, Cambridge, MA, USA). Appropriate HRP-conjugated goat anti-mouse IgG (ab97040) and anti-rabbit IgG (ab205718) secondary antibodies were also obtained from Abcam (Cambridge, MA).
4
Immunoprecipitation of CLDN4 and YAP1 Analysis
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Immunoprecipitation was performed according to the method described previously [51 (link)]. Briefly, whole-cell lysates were pre-cleaned in lysis buffer with protein A/G agarose (Santa-Cruz) for 1 h at 4 °C and subsequently centrifuged. The supernatants were incubated with antibody against CLDN4 (4D3) and protein A/G agarose for 3 h at 4 °C. Precipitates were collected by centrifugation, washed five times with lysis buffer, solubilized with sample buffer (40 µL; Sigma), and subjected to immunoblot analysis with antibodies against YAP1 (Abcam) or phosphoserine (Abcam, ab9332), or phosphotyrosine (Abcam, ab179530). Loading protein volume was confirmed by slot blot analysis of 10 µL of the samples by Coomassie blue staining (Bio-Rad, Hercules, CA, USA).
5
Phosphorylation Kinase Assay for FER
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For pFER-KD, purified FER-KD protein was incubated with 1 mM ATP and 10 mM Mg2+ for a 30-min reaction at 25°C, followed by buffer exchange into 20 mM Tris (pH 7.5) using centrifugal concentrators (Millipore, UFC901096).
For in vitro kinase assays, all samples were incubated with 1 mM ATP and 10 mM Mg2+ at 25°C for 30 min or for the specified time period. The samples were mixed with loading buffer (v/v) and boiled at 98°C for 5 min. All samples were separated by SDS–PAGE and either stained with Coomassie blue G250 or transferred to a nitrocellulose membrane (PALL, P-N66485) for protein immunoblotting using anti-phosphothreonine (Abcam, ab218195), anti-phosphoserine (Abcam, ab9332), and anti-phosphotyrosine (Abcam, ab179530) antibodies.
For the in vivo FER phosphorylation assay, anti-FERpY704 antibodies were generated by ABclonal (Wuhan, China) (Liu et al., 2022 (link)). RALF1 was synthesized by Guoping Pharmaceutical and dissolved in water. For RALF1-induced phosphorylation of FER, 7-day-old pFER::FER-GFP seedlings were treated with liquid 1/2 Murashige and Skoog medium containing 1 μM RALF1 for different times. Total protein was extracted from the seedlings and analyzed by SDS‒PAGE and immunoblotting with anti-pY704 and anti-GFP antibodies from ABclonal.
For in vitro kinase assays, all samples were incubated with 1 mM ATP and 10 mM Mg2+ at 25°C for 30 min or for the specified time period. The samples were mixed with loading buffer (v/v) and boiled at 98°C for 5 min. All samples were separated by SDS–PAGE and either stained with Coomassie blue G250 or transferred to a nitrocellulose membrane (PALL, P-N66485) for protein immunoblotting using anti-phosphothreonine (Abcam, ab218195), anti-phosphoserine (Abcam, ab9332), and anti-phosphotyrosine (Abcam, ab179530) antibodies.
For the in vivo FER phosphorylation assay, anti-FERpY704 antibodies were generated by ABclonal (Wuhan, China) (Liu et al., 2022 (link)). RALF1 was synthesized by Guoping Pharmaceutical and dissolved in water. For RALF1-induced phosphorylation of FER, 7-day-old pFER::FER-GFP seedlings were treated with liquid 1/2 Murashige and Skoog medium containing 1 μM RALF1 for different times. Total protein was extracted from the seedlings and analyzed by SDS‒PAGE and immunoblotting with anti-pY704 and anti-GFP antibodies from ABclonal.
6
Protein Extraction and Analysis
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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to GCLc (ab240379), GCLm (ab126704), Band 3 (ab108414), GAPDH (ab9485), and phosphotyrosine (ab179530) were obtained from Abcam (Cambridge, MA). Electrophoresis equipment and related supplies were obtained from Bio-Rad (USA). The spectrophotometer was purchased from Thermo Fisher Scientific (USA).
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