The anti-TRPC3/6/7 (sc-15058, 1/1,000), anti-GFP (sc-9996, 1/1,000), anti-AT1R (sc-1173, 1/1,000), anti-HA (sc-7392, 1/1,000), anti-PLCγ1 (sc-81, 1/1,000) antibodies were purchased from Santa Cruz. The anti-Flag (2368, 1/1,000), anti-GAPDH (5174, 1/1,000), anti-GST (2622, 1/1,000) antibodies were from Cell Signaling. The anti-HA-tag beads and anti-GFP-tag beads were from Medical & Biological Laboratories Co., Ltd. (Japan). The anti-Flag M2 beads (A2220) were from Sigma. The anti-β-arrestin-1 (A1CT, 1/5,000) and anti-β-arrestin-2 antibodies (A2CT, 1/2,000) were generous gifts from Dr R.J. Lefkowitz (Duke University). The pertussis toxin was from Enzo Life Sciences. The SNX482 was from Abcam. The LaCl3 and CdCl2 were purchased from Sangon Biotech (Shanghai) Co. The Fura-2-AM was from Invitrogen. The DMEM medium was from Thermo Scientific. All other chemical or reagents were purchased from Sigma.
Anti gfp sc 9996
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-GFP (sc-9996) is a primary antibody produced in mouse and specific to the green fluorescent protein (GFP). This antibody is intended for use in various immunological techniques to detect and study GFP-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag f3165, by Merck Group (4 mentions)
Anti flag, by Merck Group (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti ha sc 7392, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh sc 32233, by Santa Cruz Biotechnology (2 mentions)
Anti β actin a1978, by Merck Group (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Hybond, by Cytiva (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Recombinant human tgf β1, by Cell Signaling Technology (2 mentions)
Anti smad2 3, by Cell Signaling Technology (2 mentions)
Anti calnexin, by Cell Signaling Technology (2 mentions)
Anti phosphor smad2 3, by Cell Signaling Technology (2 mentions)
Anti hsp90, by Cell Signaling Technology (2 mentions)
Anti smad2, by Cell Signaling Technology (2 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 5174, by Cell Signaling Technology (1 mentions)
Pertussis toxin, by Enzo Life Sciences (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
34 protocols using anti gfp sc 9996
1
Antibody and Chemical Reagent Protocol
2
Immunoblot Analysis of GFP-VP26 Protein
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Cellular proteins were extracted from Vero cells using SDS sample buffer 1× (62.5 mM of Tris-HCl, pH = 6.8; Dithiothreitol (DTT) 1 M; 10% glycerol; 2% SDS; 0.01% Bromophenol Blue), and immunoblot analysis was performed using an equal quantity of proteins. The proteins were revolved on SDS 10% polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (BioRad Life Science Research, Hercules, CA, USA). The membranes were probed overnight at 4 °C with specific antibodies to detect GFP-VP26 protein. Specific proteins were detected with a secondary anti-mouse antibody linked to horseradish peroxidase (HRP). GAPDH was used as a loading control. The chemiluminescence was detected by using Western HRP substrate (Merk, Millipore, Burlington, MA, USA). Immunoblot band intensity was quantified by densitometry analysis using the TINA software (version 2.10, Raytest, Straubenhardt, Germany). Anti-GFP (sc-9996) and Anti-GAPDH (sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Goat anti-mouse immunoglobulin G (IgG) antibody-HRP conjugate was purchased from Merk, Millipore.
3
Antibody and Chemical Reagents for Cell Analysis
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The following antibodies were used: anti-Flag (F3165) and anti-β-actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-GFP (sc-9996) and anti-Lamin A/C (sc-6215) (Santa Cruz biotechnology, Dallas, TX, USA); anti-Tubulin (LF-PA0146A) (AbFrontier, Seoul, Korea); anti-PHF20 (#3934), anti-WDR5 (#13105), and anti-LC3 (#2775) (Cell Signaling Technology, Danvers, MA, USA); anti-HA (#MMS-101R) (Covance, Princeton, NJ, USA); Alexa Fluor 488 donkey anti-rabbit IgG (A21206) and Alexa Fluor 594 donkey anti-mouse IgG (A21203) (Invitrogen, Waltham, MA, USA). The following chemicals were used: hygromycin (H3274), puromycin (P8833), and CQ (C6628) (Sigma-Aldrich, St. Louis, MO, USA); Bafilomycin A1 (#11038) (Cayman, Ann Arbor, MI, USA); and rapamycin (R-5000) (LC laboratories, Woburn, MA, USA).
4
GFP-tagged Protein Purification and Western Blot Analysis
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Fifty microlitre of GFP-Trap A agarose beads (Chromotek) was washed in 500 μl buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS and 0.5% NP-40) containing a protease inhibitor cocktail. Five milligram of relevant GFP-tagged protein lysate was mixed with prewashed GFP beads and incubated overnight at 4 °C. Beads were subsequently washed on ice three times with washing buffer. For elution, the washed beads are resuspended in 100 μl of 2 × LDS sample buffer (Thermo Scientific) and boiled at 95 °C for 5 min. The eluate was loaded onto SDS page gel followed by western blot analysis using standard protocols. For western blot analysis, the following primary antibodies were used: anti-GFP (sc-9996, Santa Cruz), anti-GFP (2956, Cell Signaling), streptavidin-HRP (3999, Cell Signaling), anti-Gapdh (ab8245, Abcam) and anti-actin (ab8224, Abcam). Anti-H3K56 acetylation (39281; Activemotif), anti-K-acetylation (9441, Cell Signaling), anti-K-succinylation (PTM-401, PTM Biolabs), anti-Porin (16G9E6BC4, Life technologies, Carlsbad, CA, USA) and HRP-coupled anti-mouse or anti-rabbit secondary antibodies were from Jackson Immunoresearch. All western blots presented in the main text have been included as uncropped scans in Supplementary Fig. 6 .
5
Modulating Protein Acetylation Dynamics
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Antibodies employed were: anti-PCAF (sc-13124) from Santa Cruz (Dallas, TX, USA) used 1:50 for immunofluorescence (IF) or 1:100 for western blotting (WB); anti-lamin A/C, goat polyclonal (SC-6215) from Santa Cruz (Dallas, TX, USA) used at 1:100 dilution for and in situ proximity ligation assay (PLA); anti-HDAC2, rabbit polyclonal (AB16032) from Abcam (Cambridge, UK) used at 1:2000 for WB and 1:200 for PLA analysis; anti-H3K9 acetylated, rabbit polyclonal (07-352) from Merck Millipore (Milan, Italy) used at 1:200 for IF; anti-FLAG tag (F3165) from Sigma (St.Louis, MO, USA) and anti-HA tag (sc-7392) from Santa Cruz (Dallas, TX, USA) were used at 1:1000 in WB and 1:300 in IF; anti-GFP (sc-9996) from Santa Cruz (Dallas, TX, USA) was used at 1:500 in WB; anti acetyl-lysine (9824S) from Cell Signaling (Leiden, The Netherlands) was used at 1:2000 in WB; anti myogenin from Santa Cruz (Dallas, TX, USA) was used at 1:100 in IF.
A total of 2 μM of Mevinolin (Sigma, St. Louis, MO, USA), a drug able to induce non-farnesylated prelamin A accumulation through inhibition of farnesyl production, was added to HEK293 cells for 18 h. A total of 100 μM of Sirtinol, a SIRT1 inhibitor, able to induce PCAF acetylation [31 (link)], was added to human fibroblasts for 18 h; while 50 μM of sirtuin 1 activators (MC 2562 and MC 2528) PCAF deacetylating drugs were added for 18 h.
A total of 2 μM of Mevinolin (Sigma, St. Louis, MO, USA), a drug able to induce non-farnesylated prelamin A accumulation through inhibition of farnesyl production, was added to HEK293 cells for 18 h. A total of 100 μM of Sirtinol, a SIRT1 inhibitor, able to induce PCAF acetylation [31 (link)], was added to human fibroblasts for 18 h; while 50 μM of sirtuin 1 activators (MC 2562 and MC 2528) PCAF deacetylating drugs were added for 18 h.
6
Antibody and Drug Protocol for Cancer Research
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Antibodies used in this study were obtained from commercial sources as follows: anti-β-catenin (610153; BD Transduction Laboratories), anti-Axin2 (76G2; Cell Signaling/WB; HPA042344; Sigma/IHC), anti-GFP (sc-9996; Santa Cruz), anti-GAPDH (Epitomics), anti-CD44-APC (C26; BD Pharmigen), APC mouse IgG2b k isotype control (BD Pharmigen), anti-DCLK1 (Abcam) and PE conjugated-goat anti-rabbit IgG H&L (Abcam). EGF, bFGF, and Wnt3a were obtained from R&D systems. Verapamil, Hoechst 33342, PI, KY02111, cisplatin, and oxaliplatin were purchased from Sigma. miR-103/107 precursors and miR-103/107 LNAs (antagomirs) were purchased from Ambion and EXIQON, respectively.
7
Influenza Protein Interaction Analysis
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MG132 (133407-82-6) was purchased from APExBIO. anti-Flag M2 Affinity Gel (Flag beads, A2220), 3×Flag peptide (F4799), and chloroquine (CQ, C6628) were purchased from Sigma-Aldrich. Cycloheximide (CHX, HY-12320) was purchased from MedChemExpress (MCE). The following antibodies were used for co-Immunoprecipitation (co-IP) and western blotting: anti-Flag (F1804, Sigma), anti-α-Tubulin (PM054, MBL), anti-HA (sc-805, Santa Cruz), anti-GFP (sc-9996, Santa Cruz), anti-influenza A virus NP (A01506, GenScript), anti-phosphothreonine (9381, Cell Signaling Technology, CST). Mouse monoclonal anti-influenza A virus M1 antibody was kindly provided by Dr. Wenjun Liu (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China).
8
Western Blot Antibody Validation
9
Investigating Autophagy Regulation Mechanisms
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In this study, we utilized MG132 (Z-Leu-Leu-Leu-H (aldehyde), Peptide Institute), bafilomycin A1 (Sigma), and chloroquine (Wako). The antibodies utilized in this study were anti-NRF1 (D5B10; Cell Signaling Technology), anti-p62 (PM045; MBL), anti-S403-P-p62 (4F6; MBL), anti-ULK1 (D8H5; Cell Signaling Technology), anti-TBK1 (ab109735; Abcam), anti-GFP (sc-9996; Santa Cruz), anti-LC3B (L7543; Sigma), anti-GAPDH (6C5; Santa Cruz), anti-α-tubulin (DM1A; Sigma), anti-HA (12CA5; Sigma), and anti-Myc (sc-40; Santa Cruz) for immunoblot analyses; anti-p62 (PM066; MBL), anti-ULK1 (F-4; Santa Cruz), and anti-S172-P-TBK1 (D52C2; Cell Signaling Technology) for immunofluorescence analysis; anti-ubiquitin (clone FK2) (D058-3; MBL), anti-GABARAPL1 (D5R9Y; Cell Signaling Technology) for immunofluorescence and immunoblot analyses, anti-HA (3F10; Sigma) for immunoprecipitation; and normal rabbit anti-IgG antibody (Wako) and rabbit anti-Nrf1 polyclonal antibody (raised against mouse NRF1 residues from 292 to 741)33 (link) for ChIP assay.
10
Visualizing Autophagy in Mouse Corneal Cells
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Mouse corneal endothelial cell (MCEC) WT and KO cells were grown on glass coverslips. Using Lipofectamine 3000 (L3000001; Thermo Fisher Scientific), following manufacturer's instructions, cells were transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (#84572; Addgene). Then, 72 hours post transfection, the cells were subject to treatment in assay media for 16 hours. Following this, the cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized using 0.1% Triton X-100 for 5 minutes, and blocked using 3% BSA for 1 hour. Primary antibody incubation using 1:100 anti-GFP (#sc-9996; Santa Cruz Biotechnology, Dallas, TX, USA) and 1:100 anti-RFP (#600-401-379; Rockland Immunochemicals, Gilbertsville, PA, USA) was conducted over night at 4°C. Following secondary antibody incubation, and washes, the cells were mounted using prolong gold antifade mounting media (P36930; Thermo Fisher Scientific) and examined by Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany) using 63X oil objective.
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