IP, Ub and WB assays were carried out as previously described5 (link),9 (link) using anti-Flag (F1804; Sigma), anti-ZEB2 (H260; Santa-Cruz or from Dr. Tulchinsky), anti-GFP (3E6; Invitrogen), anti-FBXW7/hCDC4 (PA1-23468; Thermo-Scientific), anti-E-cadherin (610181; BD), anti-Vimentin (RV202; Santa-Cruz), anti-α-SMA (ab5694; Abcam), anti-β-catenin (610154; BD), anti-GSK-3β (27C10; Cell-Signalling), anti-phospho-Ser/Thr (Abcam), p-c-MycT58/S62 (Cell-Signalling), HIF-1α (EP1215Y; Abcam), MCL-1 (PA5-64688; Invitrogen), P100 (EPR4686; Abcam), KLF5 (AF3758; R&D), anti-FBXW7 antibody (ab109617; Abcam) and anti-β-actin (ab6276; Abcam) antibodies.
Anti phospho ser thr
Manufactured by Abcam
Sourced in United States
Anti-phospho-Ser/Thr is a laboratory reagent that detects phosphorylation of serine and threonine residues in proteins. It is used as a tool for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Mcl 1, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti zeb2, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by Abcam (1 mentions)
Ab109617, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Cos 7 cells, by American Type Culture Collection (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti e cadherin, by Abcam (1 mentions)
Linsitinib, by Selleck Chemicals (1 mentions)
3h labeled estrone sulfate, by PerkinElmer (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti myc agarose affinity gel, by Merck Group (1 mentions)
5 protocols using anti phospho ser thr
1
Immunoblotting Assay for Protein Analysis
2
Estrone Sulfate Uptake in COS-7 Cells
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COS-7 cells were obtained from ATCC (Manassas, VA, USA). [3H]-labeled estrone sulfate (ES) was obtained from PerkinElmer (Waltham, MA, USA). Membrane-impermeable biotinylation reagents, Sulfo-NHS-SS-biotin, streptavidin agarose beads, and protein G agarose beads were purchased from Pierce (Rockford, IL, USA). Mouse anti-myc antibody (9E10) was obtained from Roche (Indianapolis, IN, USA). Mouse anti-E-cadherin, anti-GAPDH and anti-phospho-Ser/Thr antibodies were obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated anti-mouse antibody was bought from Santa Cruz (Santa Cruz, CA, USA). Dibutyryl cyclic-AMP sodium salt (Bt2-cAMP), H89 dihydrochloride hydrate (H89), insulin-like growth factor-I human (IGF-1) and anti-myc agarose affinity gel were bought from Sigma–Aldrich (St. Louis, MO, USA). IGF-1 receptor inhibitor, linsitinib, was purchased from Selleck Chemicals (Houston, TX, USA).
3
Western Blot Analysis of Phospho-Ser/Thr and ERK
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Total protein (25 μg) from the samples was separated on a 12% (wt·vol−1) SDS–PAGE gel, transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA) and incubated with anti-phospho Ser/Thr (1:1000, Abcam, Cat# ab117253), anti-pERK (1:1000, Cell Signaling Technology, Cat# 4376) or anti-ERK (1:1000, Cell Signaling Technology, Cat# 9102) antibodies and then with a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody. Finally, blots were developed using the ECL Western blotting detection system (Amersham Bioscience, Uppsala, Sweden). A density analysis of each group was performed using ImageJ v1.8.0 software.
4
Isolation and Immunoblot Analysis of Apple Proteins
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Protein isolation from apple fruit and calli and immunoblot analysis were performed as previously described (Li et al. 2015 (link)). The protein concentration of each extract was measured using a BCA protein assay kit (Cat. no. P0012S, CWBIO). Purified recombinant MdNAC72 was used to generate a specific antibody in rabbit (Abmart, China, http://ab-mart.com.cn/ ). Anti-His (Cat. no. HT501; TransGen Biotech, China), anti-GST (Cat. no. HT601; TransGen Biotech), anti-MBP (Cat. no. HT701; TransGen Biotech, Beijing, China), anti-GFP (Cat. no. HT801; TransGen Biotech, Beijing, China), anti-FLAG (Cat. no. 14793S; Cell Signaling Technology, USA), anti-Myc (Cat. no. HT101; TransGen Biotech), anti-Ub (Cat. no. ST1200, Sigma, USA), anti-phospho-p44/42 (Cat. no. 4730; Cell Signaling Technology), anti-phosphoSer/Thr (Cat. no. ab117253, Abcam, UK), and anti-tubulin (Cat. no. M20045, Abmart) antibodies were diluted 1:1,000 with Tris-buffered saline with Tween 20 (TBST buffer, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% [v/v] Tween 20) and incubated with nitrocellulose membranes (Cat. no. S80209, Pall Corporation, USA). Secondary antibodies (goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated; Cat. no. HS201 or HS101, TransGen Biotech) were diluted to 1:3,000 in TBST.
5
Characterizing GW6a Phosphorylation Dynamics
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The wild-type (or variant) N-terminal (1–573 bp) and whole CDS of GW6a were cloned into pSuper1300-Myc to express nGW6a-Myc, nGW6aS142/T186D-Myc, nGW6aS142/T186A-Myc, and GW6a-Myc. CA-OsMAPK6 was cloned into the HindIII and SpeI sites of pSuper1300-GFP to express CA-OsMAPK6-GFP. Each of these constructs was transformed into Agrobacterium EHA105 cells and co-infiltrated into tobacco leaves as indicated. Total leave protein was extracted with buffer (50 mM Tris-MES [PH 8.0], 10 mM EDTA [PH 8.0], 0.5 M sucrose, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 1 × complete protease inhibitor cocktail) and incubated with anti-Myc Nanobody Agarose Beads (KT HEALTH) at 4 °C for 3 h. The beads were washed for three times with washing buffer (10 mM Tris-HCl [PH 7.5], 0.5 mM EDTA [PH 8.0], 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 1 × complete protease inhibitor cocktail). The samples were analyzed by SDS-PAGE with 50 μM Phos-tag and detected with anti-phospho-(Ser/Thr) (Abcam) or anti-Myc. Uncropped scans of immunoblotting results are shown in Source data .
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