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 flag
Manufactured by ABclonal
Sourced in China, United States
The Anti-FLAG product is a laboratory reagent used for the detection and purification of proteins that have been engineered to contain a FLAG tag. The FLAG tag is a short peptide sequence that can be added to proteins to facilitate their identification and isolation. The Anti-FLAG reagent is a specific antibody that binds to the FLAG tag, allowing the tagged proteins to be detected or purified from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Anti myc, by ABclonal (3 mentions)
Anti srsf1, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay reagent kit, by Thermo Fisher Scientific (2 mentions)
Anti p acc, by Cell Signaling Technology (2 mentions)
Anti acc, by Cell Signaling Technology (2 mentions)
Anti hk2, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail and phosphatase inhibitor cocktail, by Apexbio (2 mentions)
Anti aldoa, by Proteintech (2 mentions)
Anti β tubulin, by Transgene (2 mentions)
Anti ldha, by Cell Signaling Technology (2 mentions)
Anti acly, by Cell Signaling Technology (2 mentions)
Anti p acly, by Cell Signaling Technology (2 mentions)
Anti pfkfb3, by Cell Signaling Technology (2 mentions)
Anti eno2, by Proteintech (2 mentions)
Anti hadh, by Proteintech (2 mentions)
Anti ppara, by Proteintech (2 mentions)
Ecl reagents, by Smart-Lifesciences (2 mentions)
Anti gfp, by ABclonal (2 mentions)
Anti yap, by ABclonal (1 mentions)
18 protocols using anti flag
1
Investigating HSP110-YAP Interaction
2
Antibody-Based Western Blot Analysis
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WB was performed as previously described [49 (link)]. Both anti-IGF2BP3 (Cat# A4444) and anti-Flag (Cat# AE063) antibodies were purchased from Abclonal (Wuhan, China). Anti-ARHGAP11A (Cat# PA5-101840), anti-PD-L1 (Cat# M033179), and anti-β-actin (Cat# AC006) antibodies were purchased from Invitrogen, Abmart (Shanghai, China), and Sigma-Aldrich, respectively. The protein level was normalized with β-actin.
3
Identification of circRPAP2-binding proteins
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A probe targeting circRPAP2 and the anti-sense for RNA pulldown were synthesized by GenePharma, and RNA pulldown was performed using the BersinBio™ RNA pulldown Kit (BersinBio, Guangzhou, China) according to the manufacturer’s protocol. MCF-7 and MDA-MB-231 cells were used for the endogenous RNA pulldown assay, whereas 293 T cells were transfected with FLAG-tagged full-length SRSF1 or SRSF1 domain plasmids for the exogenous RNA pulldown assay. Western blotting was performed using anti-SRSF1 (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-FLAG (Abclonal, Wuhan, China) antibodies. Silver staining was carried out using the Protein Silver Stain Kit (Yeasen) following the manufacturer’s protocol. The mass spectrometry analysis of differential protein bands was conducted by IBSBio.
4
Protein Extraction and Analysis Protocol
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Proteins in cells were extracted using RIPA lysis buffer (Beyotime), and concentrations were determined using a protein assay kit (Beyotime). Protein bands were scanned using the Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA). The following antibodies were used: anti-PCNA, anti-PTK2 (Abclonal), anti-SRSF1, anti-MMP2, and anti-MMP9 (Santa Cruz Biotechnology). RIP assays were performed using the BersinBioTM RNA Immunoprecipitation (RIP) Kit (BersinBio), with anti-SRSF1 (Santa Cruz Biotechnology), anti-FLAG (Abclonal), and appropriate control IgG (BersinBio) antibodies. Subsequently, qRT-PCR and agarose gel electrophoresis assays were performed. IHC was performed using antibodies against PCNA, Ki-67, PTK2, and MMP2 (Abclonal). IF was performed using an antibody against SRSF1 (Santa Cruz Biotechnology). Images were taken using a microscope (Leica Microsystems). All assays were performed according to the manufacturer’s protocol. The catalog numbers for all antibodies used were provided in Table S2 .
5
Protein Isolation from Tomato Leaves
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The total protein was isolated from 2–4 mg of fresh leaves from the tomato plant using 10 volumes (v/w) of a protein extraction buffer containing 50 mM Tris-HCl (pH 8.0), 1.5% (w/v) dithiothreitol, 12% (w/v) sucrose (Suc), and 2% (w/v) lithium lauryl sulfate. Before SDS-PAGE separation, all samples were mixed with an equal amount of 2× urea buffer containing 10 mM Tris-HCl (pH 8.0), 10% (w/v) Suc, 2% (w/v) SDS, 1 mM EDTA, 4 mM dithiothreitol, a small amount of bromophenol blue, and 10 M urea and were electrophoresed on 14% polyacrylamide gel and electro-blotted to PVDF membranes (Bio-Rad). Samples were loaded on the same weight of fresh leaves. The Flag-fused target protein was detected using anti-FLAG (ABclonal, Wuhan, China) as the primary antibody, and for the secondary antibody, HRP goat anti-mouse IgG (ABclonal, Wuhan, China) was used.
6
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).
7
Western Blotting and Immunohistochemistry Protocol
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Western blotting was performed as described previously [17 (link), 24 (link)]. The following primary antibodies were used: anti-SERPINB2 (1:1,000) (Proteintech, Wuhan, China), anti-uPAR (1:1,000) (Abcam, Cambridge, UK), anti-uPA (1:1,000) (Abcam), anti-HDAC1 (1:1,000) (Abclonal, Wuhan, China), anti-AGO2 (1:1,000) (Abclonal), anti-GST (1:1,000) (Abcam), anti-FLAG (1:1,000) (Abclonal), and anti-TDP43 (1:1,000) (Proteintech). Immunohistochemistry was performed using antibodies against TDP43 (1:200) (Proteintech), SERPINB2 (1:200) (Proteintech), and uPAR (1:200) (Abcam), as described previously [17 (link), 24 (link)].
8
Investigating Metabolic Regulators in OSRC2 Cells
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The GFP-CTL and GFP-HMGCS2 plasmids were transfected into OSRC2 cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), and anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).
9
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).
10
Antibodies for Myogenic Lineage Analysis
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Antibodies used in this study are: anti-PAX7 (DSHB, deposited by Kawakami, A.), anti-MF20 (DSHB, deposited by Fischman, D.A.), anti-Akt (Proteintech, 10176-2-AP), anti-phosphorylated-Akt (CST, 4060), anti-eIF4E (ZENBIO, 384193), anti-phosphorylated-eIF4E (CST, 9741), anti-eIF4EBP1 (ZENBIO, R24197), anti-phosphorylated-eIF4EBP1 (CST, 2855), anti-MyoD (Santa cruz, sc-32758X), anti-MyoD(Active motif, 39991), anti-Mcm2(Abcam, ab4461), anti-Myog (Santa cruz, sc-12732), anti-ACTB (Abclonal, AC026), anti-FLAG (Abclonal, AE005), anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, A-21202), anti-mouse secondary antibody conjugated with Alexa Fluor 555 (Invitrogen, A-31570), anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, A-21206), anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (Invitrogen, A-31572), HRP anti-mouse secondary antibody (beyotime, A0216), HRP anti-rabbit secondary antibody (beyotime, A0208).
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