Horseradish peroxidase hrp conjugated secondary antibody

The cells were lysed using a radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl at pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing a protease and phosphatase inhibitor cocktail (Roche). Equal amounts of proteins were separated using SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 3% bovine serum albumin/TBST and incubated overnight with specific primary antibodies. The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Genetex) for 1 h at room temperature, and proteins were detected using an enhanced chemiluminescence kit (Millipore). Protein expression was quantified using ImageJ software. Data are expressed as the mean ± standard error (SE) and represented as fold changes relative to the control, n = 3. Details on the antibodies used are listed in Supplementary Table S1.

Cell culture reagents were purchased from Welgene (Daegu, Republic of Korea). Mouse monoclonal antibodies against cleaved poly (ADP-ribose) polymerase (PARP) were purchased from BD Biosciences (San Jose, CA, USA). Rabbit polyclonal antibodies against pAMPK (T172), AMPK, pmTOR (S2448), mTOR, p70S6K (T389), p70S6K, pS6, S6, and cleaved caspase 3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against actin and SDHA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal SDHB antibody and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Primary antibodies against SDHB and SDHC were purchased from Abcam (Cambridge, UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Antibodies against E-cadherin, N-cadherin, and Matrigel basement matrix were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against IGFBP-3, vimentin, myc, glutathione S-transferase (GST), His-probe, HA-probe, OctA-probe, IGF-1R, ubiquitin, and actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against E-cadherin, N-cadherin, and tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Fluorochrome (Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 594)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human recombinant IGFBP-3 was purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant IGFBP-3 protein used or evaluation of cellular uptake of extracellular IGFBP-3 was kindly provided by Insmed Inc. (Glen Allen, VA, USA) [22 (link)]. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from MP Biomedicals (Santa Ana, CA, USA). G418 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Crystal violet, a mouse monoclonal anti-vimentin antibody, and additional chemicals unless otherwise indicated were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Differentiated BMDC from 6-to 8-week-old C57BL/6 J mice were seeded at a density of 1 × 10⁶ cells/mL and were stimulated with 5 μg/mL LPS, 5 μM rosiglitazone (Ro; Sigma-Aldrich), or 1, 10, and 25 μg/mL sub-fraction IE3-3G1 for 24 h. After washing with PBS (phosphate buffered saline), whole-cell lysates were prepared by using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) and were then boiled in sample buffer (62.5 mM Tris–Cl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 20% glycerol, 10% 2-mercaptoethanol) at 99 ℃ for 15 min. Samples were electrophoresed on 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore) for Western blotting. The membranes were blocked for 1 h with 10% non-fat milk in TBST (150 mM NaCl, 50 mM Tris–Cl [pH 7.4], 0.05% Tween 20), and incubated overnight with PPARγ (Cayman Chemical, Ann Arbor, MI, USA) and β-actin (GeneTex, Irvine, CA, USA) antibodies. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (GeneTex) for 2 h, further treated with the Western lightning chemiluminescence reagent (PerkinElmer, Waltham, MA, USA), and visualized with X-ray film. The density of detected proteins was calculated in comparison with the density of β-actin, and the results are reported as the percentage.

Total protein of leaf samples was extracted with Culture Cell Lysis Reagent (CCLR) buffer (100 mM K₂HPO₄, 100 mM KH₂PO₄, pH 7.8, containing 1 % Triton X-100, 10 % glycerol, 1 mM EDTA, and 7 mM 2-mercaptoethanol). The protein amount was measured by using the Bio-Rad Protein Assay Kit with bovine serum albumin as a standard. For western blot analysis, 30 µg total protein from each sample was loaded and separated by SDS-PAGE with a 10 % acrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membrane for antibody probing. The following primary antibodies were used: anti-eGFP, rabbit polyclonal antibody (Yao-Hong Biotechnology, Cat#YH-80005) at 1:10,000 dilution and anti-Actin mouse monoclonal antibody (Sigma, A0480) at 1:2500 dilution. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from GeneTex. Secondary anti-rabbit antibody was diluted at 1:10,000; anti-mouse antibody was diluted at 1:12,000.

BAF was obtained from Enzo Life Sciences. Cell culture reagents were purchased from Invitrogen, Sigma Lonza and Pan Biotech. PhosSTOP phosphatase inhibitor and complete EDTA-free protease inhibitor were purchased from Roche Applied Science. RIPA buffer was obtained from Millipore. Antibodies were against GAPDH (Santa Cruz, #sc-25778), 4E-BP1 (Cell Signaling, #9452) and Phospho-4E-BP1 (Thr37/46) (Cell Signaling, #9459). Horseradish peroxidase (HRP)-conjugated secondary antibodies obtained from Genetex.

Antibodies against pAkt (S473), Akt, pSrc (Y416), Src, pMEK (S217/221), MEK, Nanog, and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved PARP and HIF-1α and Matrigel were purchased from BD Biosciences (San Jose, CA, USA). Primary antibodies against 6x-His tag, Ubiquitin, and Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HSP70, HSP90, and Hop were purchased from Enzo Life Science (Farmingdale, NY, USA). Antibodies against Oct4 and Sox2 were purchased from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Ni-NTA agarose and fluorescence (Alexa Fluor 488 and Alexa Fluor 594)-conjugated secondary antibodies was purchased from Thermo Fisher Scientific. Biotinylated secondary antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). ATP-agarose was acquired from Innova Biosciences (Cambridge, UK). Propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The detailed information on used primary and secondary antibodies, including vendor, catalogue number, application, and dilution ratio (or concentration) is listed in Table S2.