IF and IHC were performed according to the conventional protocols. The primary antibodies used for IF were anti-YAP (Abcam, Hong Kong, China #ab52771) and anti-βTrCP (Abcam, #ab233638). The primary antibodies used for IHC were: anti-YAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-101199) and anti-ISG15 (Abcam, #ab233071). For IB, the proteins were resolved on SDS-PAGE gels according to the conventional protocols. The primary antibodies used were anti-ISG15 (Abcam, #ab233071), anti-YAP (Abcam, #ab52771 and Santa Cruz, #sc-101199), anti-GAPDH (CST, #5174 and #51332), anti-Ub (Abcam, #ab7780 and #ab7254), anti-PSMB5 (Abcam, #ab167341), anti-βTrCP (Abcam, #ab71753 and #ab233638), anti-YAPO241 (developed by Biolynx, Hangzhou, China), anti-YAPP127 (Abcam, #ab76252), anti-YAPP397 (CST, Boston, MA, USA, #13619), anti-HA (Abcam, #ab9110 and #ab18181), anti-TEAD4 (Abcam, #ab197589 and #ab58310), anti-6PGL (Abcam, #ab229872), anti-FLAG (CST, #8146 and #2368), anti-UbCH8 (Abcam, #ab177485), anti-HERC5 (Invitrogen, Carbsland, CA, USA, #703675), anti-ATG5 (Abcam, #ab221604), anti-LATS1 (Abcam, #ab243656), anti-CK1 (Abcam, #ab270997 and #ab115293), anti-SMAD2 (Abcam, #ab40855), anti-alpha fetoprotein (AFP, Abcam, #ab284388) and anti-albumin (Alb, Abcam, #ab207327). ELISA kits (Yingxin, Shanghai, China) were used to measure the concentration of YAP protein and Rib-5-P.
Anti ub
Manufactured by Abcam
Sourced in China
Anti-Ub is a lab equipment product that detects and binds to ubiquitin, a small regulatory protein found in eukaryotic cells. It is used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Anti mmp2, by Abcam (2 mentions)
Enhanced chemiluminescence system, by Cytiva (2 mentions)
Anti mmp9, by Abcam (2 mentions)
Anti b56γ2, by Abcam (2 mentions)
Anti ubr5, by Abcam (2 mentions)
Anti hif1a, by Abcam (2 mentions)
Anti k63 ub, by Merck Group (2 mentions)
Anti plk1, by Abcam (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti mklp1, by Abcam (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Peroxidase conjugated goat anti human igg f ab 2 fragment specific, by Jackson ImmunoResearch (2 mentions)
Anti c myc, by GenScript (2 mentions)
Anti vcp, by Santa Cruz Biotechnology (2 mentions)
Anti incenp, by Abcam (2 mentions)
Anti yap, by Abcam (1 mentions)
Anti ha, by Abcam (1 mentions)
Anti smad2, by Abcam (1 mentions)
Anti βtrcp, by Abcam (1 mentions)
Anti isg15, by Abcam (1 mentions)
8 protocols using anti ub
1
Comprehensive Protein Analysis Protocol
2
Western Blotting of DNA Damage Markers
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Western blotting was performed as previously described26 (link). The primary antibodies used in this study are as follow: anti-ATM (1:1000, CST), anti-p-ATM (S1981) (1:1000, CST), anti-H2AX (1:1000, CST), anti-γ-H2AX (S391) (1:1000, CST), anti-GAPDH (1:1000, CST), anti-PFKP (1:1000, CST), anti-B56γ2 (1:1000 Abcam), anti-CS (1:1000, CST), anti-E-Cadherin (1:1000, CST), anti-Vimentin (1:1000, CST), anti-ERK (1:1000, CST), anti-p-ERK (1:1000, CST), anti-AKT (1:1000, CST), anti-p-AKT (1:1000, CST), anti-Ub (1:2000, Abcam), anti-UbR5 (1:1000, Abcam), anti-HIF1A (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), and anti-MMP9 (1:1000, Abcam). The proteins were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). All experiments were performed at least three times.
3
In Vitro Ubiquitination of AtHUB2
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Rabbit E1 (Boston Biochem, 200 ng), purified Arabidopsis E2 AtUBC1‐His (500 ng), purified GST‐AtHUB2 (1 mg) and recombinant plant ubiquitin (Boston Biochem, 80 μg) were used for the in vitro ubiquitination assay. An AtHUB2 protein carrying two point mutations (C345A & H347A) was used as the negative control for fully functional AtHUB2. Reactions were performed at 37 °C for 2 h and then stopped by the addition of an equal volume of loading buffer (40 mm Tris‐HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% Coomassie Brilliant Blue, 0.1% bromophenol blue and 1% β‐mercaptoethanol) and boiling for 10 min.
For total plant protein extraction, leaves were ground in liquid nitrogen and dissolved in denaturing buffer containing 50 mm Tris‐HCl, pH 7.5, 150 mm NaCl, 0.1% NP‐40, 4 m carbamide and 1 mm PMSF. Protein concentrations were determined using a Coomassie protein assay kit (Bradford). Each total protein sample was analysed by 10% SDS‐PAGE and immunoblotting using relevant antibodies. The specific antibodies employed in these assays were as follows: anti‐H3 (Cell Signaling Technology); anti‐H3K4me2 (Millipore); anti‐H3K4me3 (Millipore); anti‐H3K9me2 (Millipore); anti‐H3K9me3 (Millipore); anti‐ub (abcam); and anti‐GST (Cell Signaling Technology).
For total plant protein extraction, leaves were ground in liquid nitrogen and dissolved in denaturing buffer containing 50 m
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted with RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and Halt Protease and Phosphatase Inhibitor Cocktail. Membrane protein was extracted by membrane and cytosol protein extraction kit (Beyotime Biotechnology, China). The concentration of proteins was tested using the bicinchoninic acid (BCA) protein assay. Protein samples (30 μg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Then, membranes were incubated with primary antibody at 4 °C overnight. Anti-MTPα, anti-IRS1/P-IRS1, anti-Glut4, anti-β-actin, anti-GAPDH, anti-SIRT1, anti-Ace, anti-Ub and anti- Na+-ATPase α-1 were from Abcam. Anti-Akt/P-Akt, anti-VDAC1, anti-H2B and secondary antibody were from Cell Signaling Technology. Probed membranes were washed several times with TBST, and then incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 h. Bound antibody was detected with enhanced chemiluminescence (Millipore, Billerica, MA, USA). Protein expression was quantified using Image J software (NIH, USA). Total protein expression was normalized with respect to β-actin/GAPDH expression and membrane protein was normalized with respect to Na+-ATPase α-1 expression.
5
Western Blotting of DNA Damage Markers
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Western blotting was performed as previously described26 (link). The primary antibodies used in this study are as follow: anti-ATM (1:1000, CST), anti-p-ATM (S1981) (1:1000, CST), anti-H2AX (1:1000, CST), anti-γ-H2AX (S391) (1:1000, CST), anti-GAPDH (1:1000, CST), anti-PFKP (1:1000, CST), anti-B56γ2 (1:1000 Abcam), anti-CS (1:1000, CST), anti-E-Cadherin (1:1000, CST), anti-Vimentin (1:1000, CST), anti-ERK (1:1000, CST), anti-p-ERK (1:1000, CST), anti-AKT (1:1000, CST), anti-p-AKT (1:1000, CST), anti-Ub (1:2000, Abcam), anti-UbR5 (1:1000, Abcam), anti-HIF1A (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), and anti-MMP9 (1:1000, Abcam). The proteins were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). All experiments were performed at least three times.
6
Western Blot Protein Detection
7
Western Blot Antibody Validation
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Western blots were performed using the standard protocol. Peroxidase-conjugated goat antihuman IgG, F(ab)2 fragment specific (Jackson ImmunoResearch, 109-036-006) was used as the secondary antibody (1:5000) when using sAB-K29 as the primary binder (0.2-2 μg/mL).
Other primary antibodies are diluted as followed: Anti K63-Ub (Sigma,1:1000); Anti Ub (Abcam, 1:1000); Anti β-Actin (Santa Cruz, 1:1000), Anti GAPDH (Santa Cruz, 1:2000), Anti c-Myc (Genscript, 0.3 μg/mL), Anti VCP (Santa Cruz, 1:1000), Anti INCENP (Abcam,1:3000), Anti MKLP1 (Abcam,1:1000), Anti TBK1 (CST,1:1000), Anti PLK1 (Abcam, 1 μg/mL).
Other primary antibodies are diluted as followed: Anti K63-Ub (Sigma,1:1000); Anti Ub (Abcam, 1:1000); Anti β-Actin (Santa Cruz, 1:1000), Anti GAPDH (Santa Cruz, 1:2000), Anti c-Myc (Genscript, 0.3 μg/mL), Anti VCP (Santa Cruz, 1:1000), Anti INCENP (Abcam,1:3000), Anti MKLP1 (Abcam,1:1000), Anti TBK1 (CST,1:1000), Anti PLK1 (Abcam, 1 μg/mL).
8
Protein Expression Analysis Protocol
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Cell lysates were separated using 4-12% Bis-Tris gels (Life Technologies, USA) and transferred to a PVDF membrane. The cell membrane was sealed with 5% skimmed milk in TBST buffer, and incubated with the corresponding antibody. The antibodies used were anti-RBCK1 (Abcam), GLUT1 (Abcam), PPARγ (Abcam), PGC1α (Abcam), β-catenin (CST), anti-UB (Abcam), and anti-tubulin (Santa Cruz), all at a 1:1000 dilution.
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