Recombinant Tat101 was purchased from ImmunoDiagnostics. Recombinant PDGF-BB was purchased from R&D Systems. Jagged-1 and DAPT were purchased from Sigma. Antibodies were obtained from the following sources: Ki67 (abcam), NICD (cell signaling), Actin (sigma), α-SM, Notch3 (cell signaling), VEGF-A (abcam); goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology. Flag-tagged CSL-VP16 plasmids were obtained from Dr. Aly Karsan (University of British Columbia, Vancouver, Canada) and control Notch3 siRNA (sc-29798), RBPJ siRNA (sc-41446), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology.
Goat anti rabbit sc 2004
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-rabbit (sc-2004) is a secondary antibody product designed for use in various immunological techniques. It is produced by immunizing goats with rabbit IgG and purifying the resulting antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse sc 2005, by Santa Cruz Biotechnology (11 mentions)
Notch3, by Abcam (1 mentions)
Vegfa, by Abcam (1 mentions)
Recombinant pdgf bb, by R&D Systems (1 mentions)
Jagged1, by Merck Group (1 mentions)
Scrambled sirna sc 37007, by Santa Cruz Biotechnology (1 mentions)
Tfeb a303 673a, by Fortis Life Sciences (1 mentions)
Bafilomycin a1 b1793, by Merck Group (1 mentions)
Rapamycin r8781, by Merck Group (1 mentions)
Ctsd ab75852, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Np 40, by Beyotime (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti ha 6e2, by Cell Signaling Technology (1 mentions)
Immobilon western chemiluminescent hrp substrate ecl working solution, by Merck Group (1 mentions)
Anti hcv core c7 50, by Abcam (1 mentions)
21 protocols using goat anti rabbit sc 2004
1
Notch Signaling in Vascular Remodeling
2
Western Blot Quantification Protocol
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The blots were probed as outlined in Supplemental Table 3 . Secondary antibodies, goat anti-mouse (sc-2005) and goat anti-rabbit (sc-2004), were from Santa Cruz Biotechnologies (Paso Robles, CA). Bound antibody was detected with horseradish peroxidase-conjugated secondary antibody and chemiluminescence. Bands were quantified using ImageJ software from the National Center for Biotechnology Information (available at: http://rsbweb.nih.gov/ij/ ). The images were converted into binary mode, and ratios were derived by comparing the protein of interest bands to β-actin.
3
Autophagy Marker Antibody Validation
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Antiretroviral drugs TDF and FTC (Gilead Sciences, Foster City, CA, USA), and DTG (ViiV Healthcare, Research Triangle Park, NC, USA). Rapamycin (R8781) and bafilomycin A1 (B1793) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Antibody resources: BECN1 (sc-11427) and CTSB (sc-365558) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA. LAMP2 (NB300-591) and MAP1LC3B (NB100-2220) were purchased from Novus Biological Company, Centennial, CO, USA. CTSD (ab75852) and M6PR (ab124767) were purchased from Abcam, Cambridge, MA, USA. TFEB (A303-673A) was purchased from Bethyl Laboratories, Montgomery, TX, USA. SQSTM1 (MBL PM045) was purchased from MBL International, Woburn, MA, USA. Goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.
4
EMT and Cell Cycle Protein Expression
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Protein expression in the process of EMT and cell cycle were evaluated by western blot analysis. Briefly, total proteins from ME180 and C33A cells were collected by lysis buffer (NP-40; Beyotime, Nantong, China) on ice and quantified using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (50 µg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred to a nitrocellulose membrane (NC, Millipore, MA, USA). The membrane was blocked for 1 h with 5% skimmed milk at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibodies against Cyclin B1 (sc-70898, 1:1,000), CDC25C (sc-327, 1:1,000), E-Cadherin (sc-71009, 1:1,000), N-Cadherin (sc-53488, 1:1,000) and GAPDH (sc-32233, 1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). After washing with TBST for 4 times, the membrane was then incubated with secondary goat-anti-rabbit (sc-2004) or goat-anti-mouse (sc-2005) antibody (Santa Cruz Biotechnology, Inc.) for 1 h at 37°C with a dilution of 1:1,000. Finally, the proteins were quantified using ECL Prime Western Blotting Detection reagent (GE Healthcare, Parsippany, NJ, USA) and an ImageQuant LAS 4000 Mini Biomolecular Imager (GE Healthcare).
5
Immunoblotting for HCV Proteins
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Proteins extracted from cells or concentrated from elutriants were analyzed with SDS-PAGE, the proteins on the membrane were probed with anti-HCV Core (C7-50, Abcam Ltd.), anti-NS3 (H23, Abcam Ltd.), anti-hA3G (ab75560, Abcam Ltd.), anti-HA (6E2, Cell Signaling Biotechnology Inc.), or anti-HA-tag [HRP] (A00169, GenScript.) antibody, respectively, with anti-Actin antibody (TA-09, ZSGB-BIO, China) served as the control. After washing with TBST, the membrane was incubated with goat anti-mouse (sc-2005, Santa Cruz Biotechnology Inc.) or goat anti-rabbit (sc-2004, Santa Cruz Biotechnology Inc.) secondary antibody, respectively. Protein signals were visualized and captured using Immobilon Western Chemiluminescent HRP Substrate ECL working solution (Millipore Inc.) with ChemiDo XRS gel imager system (Bio-Rad, CA).
6
Western Blot Analysis of Cell Cycle Proteins
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SDS-PAGE and Immunoblotting was performed as described previously (Lee, 2012 (link)).
For immunoblotting analysis the following commercial primary antibodies were used
in this study: anti-Cyclin D1 (#2922) and anti-Cyclin D3 (# 2936) were purchased
from Cell Signaling Technology (Danvers, MA, USA); anti-Cyclin B1 (H-433),
anti-p53 (SC-126), and anti-ERK1 (SC-94) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA); anti-p21 (ab7960) was purchased from Abcam
(Cambridge, MA, USA). The primary antibodies were visualized with goat
anti-rabbit (SC-2004, Santa Cruz Biotechnology, Santa Cruz) or goat anti- mouse
(SC-2005, Santa Cruz Biotechnology) antibodies conjugated with horseradish
peroxidase and an enhanced chemiluminescence (ECL) detection system.
For immunoblotting analysis the following commercial primary antibodies were used
in this study: anti-Cyclin D1 (#2922) and anti-Cyclin D3 (# 2936) were purchased
from Cell Signaling Technology (Danvers, MA, USA); anti-Cyclin B1 (H-433),
anti-p53 (SC-126), and anti-ERK1 (SC-94) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA); anti-p21 (ab7960) was purchased from Abcam
(Cambridge, MA, USA). The primary antibodies were visualized with goat
anti-rabbit (SC-2004, Santa Cruz Biotechnology, Santa Cruz) or goat anti- mouse
(SC-2005, Santa Cruz Biotechnology) antibodies conjugated with horseradish
peroxidase and an enhanced chemiluminescence (ECL) detection system.
7
Antiretroviral Drugs and Oxidative Stress
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Antiretroviral drugs TDF, FTC (Gilead Sciences, Foster City, CA, USA), and DTG (ViiV Healthcare, Research Triangle Park, NC, USA) were used. NAC (A7250) and (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO; SML0737) were purchased from Sigma-Aldrich, St. Louis, MO, USA, and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL; sc-200825) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Antibody resources: beclin 1 (BECN1; sc-11427) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Lysosome associated membrane protein 2 (LAMP2; NB300-591), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; NB100-2220), and integrin subunit alpha M (ITGAM; NB110-89474) were purchased from Novus Biological Company, Centennial, CO, USA. Cathepsin D (CTSD; ab75852) and caspase 3 (CASP3; ab13585) were purchased from Abcam, Cambridge, MA, USA. Galectin 3 (GAL3) (A3A12) was purchased from Invitrogen. Sequestosome 1 (SQSTM1; PM045) was purchased from MBL International, Woburn, MA, USA, and allograft inflammatory factor 1 (AIF1; 019-19741) was purchased from Wako Pure Chemicals Industries, Chuo-ku, Osaka, Japan. Goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.
8
Western Blotting Protocol for Protein Analysis
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The standardized protocol [24] was adopted for Western Blotting. Specific primary antibodies- Syp (Santa Cruz Biotechnology. sc9116); PSD95 (Pierce Biotechnology, Inc. MA1045) and loading control (GAPDH & β actin; Bioss, USA) in dilutions of 1:1000 were used for primary incubation followed by overnight incubation with secondary antibody (Goat anti mouse sc 2005 or Goat anti rabbit sc 2004, SantaCruz Biotechnology; dilution- 1:2000). Bands were visualized using DAB. The blots were scanned for densitometric analysis using Quantity 1 software of Gel Documentation System (Bio-Rad, USA). The result was expressed as change in terms of control as follows: where, ODPE, LE, PC, LC referred to OD of experimental group, loading control in experimental group; control group; loading control in control group respectively.
9
Western Blot Analysis of Metabolic Proteins
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Cells were washed with phosphate-buffered saline (PBS) and lysed with sodium dodecyl sulfate (SDS) lysis buffer [60 mM Tris-HCl, pH 6.8, 1 % SDS in distilled water (DW)] containing protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein from each sample were separated by SDS-polyacrylamide gel electrophoresis on 8%–12% gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with primary antibodies against monocarboyxlate transporters: MCT1 (AB3538P; Millipore), MCT2 and MCT4 (SC50322 and SC50329; Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl Co-A synthase 2 (SC85258; Santa Cruz Biotechnology), fatty acid synthase (3189; Cell Signaling Technology, Danvers, MA, USA), and actin (A1978; Sigma-Aldrich, St. Louis, MO, USA). Membranes were washed in PBS and incubated with goat anti-rabbit (sc2004) or anti-mouse (sc2005) IgG horseradish peroxidase (Santa Cruz Biotechnology) as the secondary antibody. Labeled, specific protein bands were visualized using the ECL Kit (Thermo Scientific, Waltham, MA, USA).
10
Western Blotting Protocol for Protein Analysis
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Total protein extract was obtained by homogenizing tissue in RIPA buffer (PBS, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5.5% β-glycerophosphate, 1 mM dithiothreitol and complete protease and phosphatase inhibitors (Roche Diagnostics, Vilvoorde, Belgium)). The total protein yield was determined using Bradford reagent (Biorad, Temse, Belgium) and 30 µg protein was fractionated by SDS-PAGE. Protein lysates from multiple mice per group were pooled and transferred to a nitrocellulose membrane which was subsequently blocked for 1 h with 5% BSA, incubated overnight at 4 °C with primary antibodies in blocking buffer (Table S2 ), followed by 1 h incubation at room temperature with horse radish peroxidase-conjugated secondary antibodies (goat-anti rabbit sc2004, Santa Cruz, Heidelberg, Germany or goat anti-mouse sc2005, Santa Cruz). Clarity western ECL substrate (Biorad, Temse, Belgium) was used to visualize the proteins. GAPDH and β-tubulin were used as loading control proteins or total protein load was used for normalization.
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