To verify the potential direct interaction between HSP110 and YAP, HEK293T cells were co-transfected with pECMV-3-FLAG-N-HSP110 (HSP110-Flag) alone or together with PCMV-MYC-YAP (YAP-Myc) for 48 h. The potential interaction between HSP110 and YAP was precipitated using anti-Myc (IP, Abclonal) and detected using anti-Flag (Abclonal) and anti-Myc (Abclonal) by western blot. Moreover, the interaction between YAP and TEAD4 in HPAMSCs was verified using anti-YAP (IP, Santa cruz) and the precipitates were detected by western blot using anti-TEAD4 (abclonal).
Anti myc
Manufactured by ABclonal
Sourced in China, United States
The Anti-Myc is a laboratory equipment product designed for the detection and purification of proteins tagged with the c-Myc epitope. It is a highly specific and sensitive antibody that recognizes the c-Myc tag, which is commonly used in various protein expression and purification systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by ABclonal (3 mentions)
Anti β actin, by ABclonal (2 mentions)
Anti gst, by ABclonal (2 mentions)
Anti gfp, by Transgene (2 mentions)
Myc trap magnetic agarose beads, by Proteintech (2 mentions)
Ae010, by Transgene (2 mentions)
Ht801 02, by Transgene (2 mentions)
Anti gfp, by ABclonal (2 mentions)
Anti yap, by ABclonal (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
A2220, by Merck Group (1 mentions)
N7885, by Merck Group (1 mentions)
T1426, by Merck Group (1 mentions)
Ac026, by ABclonal (1 mentions)
H6908, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Ae003, by ABclonal (1 mentions)
10 protocols using anti myc
1
Investigating HSP110-YAP Interaction
2
Antibody-based Target Identification Workflow
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The antibodies used in this study were as follows: anti-C1QTNF5, anti-Lamin B1, anti-Na+/K+-ATPase, anti-Myc, anti-β-Actin, anti-GAPDH, anti-GST and anti-His (A3021, A1910, A11683, AE070, AC026, AC002, AE001 and AE003, respectively; ABclonal, China); anti-α-Tubulin, anti-Flag and anti-HA (T6199, F1804 and H6908, respectively; Sigma); anti-NP (ab128193; Abcam); anti-H1N1 HA (11684-MM03; Sino Biological); goat anti-mouse or anti-rabbit IgG (115-035-174 and 111-005-144; Jackson Immuno Research Laboratories); goat anti-mouse antibodies conjugated with Alexa Fluor 488 (A-11001; Invitrogen). The main reagents used in this study were as follows: anti-Flag M2 beads, TPCK, sialidase and blasticidin (A2220, T1426, N7885 and 203350, respectively; Sigma); Lipofectamine 2000 (11668019; Thermo Fisher Scientific); Dual luciferase reporter assay kit (E1980; Promega); Neofect™ DNA transfection reagent (TF20121201; Neofect); Minute™ plasma membrane protein and cell component separation kit (SM-005; Invent Biotechnologies).
3
Protein Interaction Profiling in Arabidopsis
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The ZmWEE1, ZmPP2Ac‐2, and ZmTE1 genes were cloned into the pCAMBIA1390‐7Myc‐6HIS or pEareyGate 101‐YFP vectors to generate the 35S::ZmTE1‐Myc, 35S::ZmTE1‐YFP, 35S::ZmPP2Ac‐2‐Myc, and 35S::ZmWEE1‐YFP plasmid, respectively. Constructs were then transformed into Arabidopsis mesophyll cells for transient protein expression. Co‐immunoprecipitation (Co‐IP) was performed according to a previous study (Lv et al., 2020 (link)). In brief, Arabidopsis mesophyll cells were harvested and lysed in cell lysis buffer (0.5 mm EDTA; 10 mm Tris‐HCl; pH 7.5; 0.5% NP‐40; 1 mm PMSF; 150 mm NaCl) on ice for 30 minutes with pipetting every 10 minutes. Cell lysates were centrifuged, and the supernatant was incubated with MYC‐Trap magnetic agarose beads (Chromotek, catalog number ytma20, Germany) at 4 °C for 2 h. The beads were washed three times with dilution buffer (10 mm Tris‐HCl; pH 7.5; 150 mm NaCl; 0.5 mm EDTA) and then re‐suspended in SDS loading buffer. The re‐suspended beads were boiled for 10 minutes followed by western blotting using anti‐MYC (Abclonal, catalog number AE010, Wuhan, China) or anti‐GFP (TransGen Biotech, catalog number HT801‐02, Beijing, China) antibody.
4
Western Blot Protein Detection Protocol
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Proteins were separated on SDS‐PAGE gels (10%), before being transferred to Immune‐Blot PVDF membranes (Roche). Subsequently, the membranes were incubated in blocking buffer for 2 h at 4°C. After blocking, the membranes were incubated with anti‐His (#AE003, ABclonal), anti‐GFP (#AE011, ABclonal), anti‐GST (#AE006, ABclonal), anti‐MBP (#AE016, ABclonal), anti‐myc (#AE009, ABclonal), anti‐actin (#AC009, ABclonal) antibodies for 2 h at 4°C. The membranes were subsequently washed with blocking buffer for three times, before being incubated with the secondary antibody HRP goat anti‐rabbit IgG (H + L) antibody (#AS014, ABclonal), or HRP goat anti‐mouse IgG (H + L) antibody (#AS003, ABclonal). Finally, the membranes were washed with TBST and they were developed using the eECL western blot kit (CWBIO, Beijing, China).
5
MYC-Trap Immunoprecipitation and Western Blot
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Cells were harvested and lysed in cell lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and 1 mM PMSF) on ice for 30 min with pipetting every 10 min. Cell lysate was centrifuged and the supernatant was incubated with MYC-Trap magnetic agarose beads (Chromotek, catalog number ytma-20, Germany) at 4°C for 2 h. The beads were washed three times with dilution buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM EDTA) and resuspended in SDS loading buffer. The resuspended beads were boiled for 10 min at 99°C and western blotting was followed using anti-MYC (Abclonal, catalog number AE010, MA, United States) or anti-GFP (TransGen Biotech, catalog number HT801-02, Beijing, China) antibody.
6
Co-Immunoprecipitation of Cellular Proteins
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The HEK293T or BHK-21 cells seeded in 6 well plates were transfected with the indicated plasmid combinations using Lipofectamine 2000 reagent (Invitrogen, MA, USA). At 24 h post transfection, the cells were harvested and lysed, and the cell extracts were subjected to Western blot analysis and co-immunoprecipitation. Briefly, the cell extracts were incubated with either mouse IgG or anti-myc (ABclonal, MA, USA, AE010, 1:100 dilution) antibody overnight at 4 °C with continuous rotation, and then the protein A/G agarose beads (Santa Cruz, CA, USA) were added to the mixtures and incubated for 6 h with continuous rotation. After incubation, the beads were collected via centrifugation and washed 5 times. The input cell extracts and immunoprecipitates were then detected by Western blotting with rabbit anti-desmin (Proteintech, IL, USA, 16520-1-AP, 1:10,000 dilution), rabbit anti-RABV-M (LSBio, WA, USA, LS-C369074, 1:2000 dilution) antisera, mouse anti-flag (ABclonal, MA, USA, AE004, 1:2000 dilution), or mouse anti-β-actin (Cwbio, Jiangsu, China, CW0096, 1:2000 dilution) monoclonal antibodies.
7
Comprehensive Protein Marker Analysis
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Anti-Flag (F-3165, 1:5000, Sigma), anti-HA (SC-7392, 1:2000, Santa Cruz Biotechnology), anti-MYC (AE010, 1:1000, Abclonal Technology), Anti-TUBULIN (SC-23948, 1:10000, Santa Cruz Biotechnology). Anti-Aggrecan (13880-1-AP,1:1000, Proteintech); Anti-MMP13 (18165-1-AP, 1:1000, Abcam); Anti-ADAMTS5 (PA5-14350, 1:1000, Thermo); Anti-ZMPSTE24(A-8858, IHC 1:50, ABclonal). Anti-H3K27me1 (2500674, 1:1000, Millipore), Anti-H3K27me2 (ab24684, 1:1000, Abcam), Anti-H3K27me3 (07-449, 1:1000, Millipore), anti-EZH2 (5246, 1:1000, CST), anti-Suz12 (sc-271325, 1:1000, Santa Cruz), anti-EED (2514035, 1:1000, Millipore).
8
Co-immunoprecipitation of RhHDA6-GFP and ARF18-MYC
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For co-immunoprecipitation assays, 35S::RhHDA6-GFP was co-infiltrated with 35S::ARF18-MYC into N. benthamiana leaves, and co-infiltration of 35S::GFP and 35S::ARF18-MYC was used as a negative control. Total proteins were extracted from N. benthamiana leaves 3 d after infiltration using extraction buffer [50 mM Tris–HCl, pH 7.5, 5 mM EGTA, 10 mM Na3VO4, 10 mM NaF, 50 mM β-mercaptoethanol, 10 mM DTT, 1 mM PMSF, 5% (v/v) glycerinum and 1% (v/v) protease inhibitor cocktail (Sigma, St Louis, USA)]. The supernatant with different protein combinations was incubated with anti-MYC antibody (Sigma, St Louis, USA) and then analyzed by western blotting using anti-MYC (Abclonal, Wuhan, China) and anti-GFP (Abmart, Shanghai, China) antibodies. The primers used for co-immunoprecipitation assays are listed in Supplemental Table S6 .
9
Antibody and Reagents Utilized for Research
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The following antibodies were used throughout this study: from ABclonal(Wuhan, China), anti-FLAG (AE063; 1:2500 for Western blot), anti-Myc (AE070; 1:2500 for Western blot), anti-β-Actin (AC026; 1:5000 for Western blot). From Abcam (Cambridge, UK), anti-hnRNP U (ab264142; 1:1000 for Western blot). From Proteintech (Wuhan, China), HRP goat anti-mouse IgG (15014; 1:5000 for Western blot), HRP goat anti-rabbite IgG (15015; 1:5000 for Western blot). anti-FLAG Magnetic Beads (HY-K0207) was purchased from MCE. 2 × Taq Master Mix (P112), High fidelity PCR enzyme- 2 × Phanta Max Master Mix (P515) were purchased from Vazyme (Nanjing, China). PMD18-T (6011) was purchased from Takara (Beijing, China). Cell Genome Extraction Kit (DP304), Plasmid Extraction Kit (DP103, DP108, DP117) were purchased from Tiangen (Beijing, China). Gel Extraction Kit (CW2302) was purchased from CWBIO (Nanjing, China). Luciferase detection kit (E6110) was purchased from Promega (Madison, USA). Cell Counting Kit (CCK-8) and TUNEL-Alexa Flour 640 apoptosis kit were purchased from Yeasen (Shanghai, China). Luciferase and nanoluc detection kit (E6110, N1110) was purchased from Promega (Madison, USA). Recombinant human IFN-alpha (11200-1), recombinant human IFN-beta (8499-IF) and recombinant human IFN-gamma (285-IF) were purchased from R&D Systems (UN).
10
Western Blot Analysis of Protein Expression
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Proteins were separated by SDS-PAGE and transferred from the gel to a PVDF membrane (Roche) in transfer buffer (25 mM Tris, 200 mM glycine, and 20% methanol). The membrane was then blocked in TBST buffer (Tris-buffered saline with 0.05% Tween 20 [pH 7.2]) containing 10% non-fat dry milk under gentle shaking. The blocked membrane was incubated with specific antibodies dissolved in TBSTM (TBST with 5% non-fat dry milk) at a ratio of 1:2000 and incubated at 4 C with shaking at 50 rpm overnight, followed by three washes (10 min each) with TBST. Next, the membrane was incubated with a secondary antibody coupled with horseradish peroxidase (HRP), which was also dissolved in TBSTM at a ratio of 1:2000, at room temperature for 1.5 h with shaking. Thereafter the membrane was washed three times (10 min each) with TBST and one time with TBS, then incubated with ECL (#CW0049S, ComWin) before photographing using a molecular imager (ChemiDoc XRS+, Bio-Rad). The primary antibodies used in our experiments include anti-FLAG (#AE005, ABclonal), anti-GFP (#AE012, ABclonal), anti-myc (#AE010, ABclonal), and anti-HA (#HT301-01, Transgen). The secondary antibodies include HRP goat anti-mouse immunoglobulin G (IgG) (H + L) antibody (#AS013, ABclonal) and HRP goat anti-rabbit IgG (H + L) antibody (#AS014, ABclonal).
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