RIN-m5F cells were lysed and centrifuged and proteins were extracted according to the manufacturer’s protocol (Sigma). The Bradford assay (Bio-Rad Laboratories) was used to quantify the protein concentrations in the supernatants. Equal amounts of protein (30 µg) from the cell extracts were separated on the pre-cast tris-glycine gel (Precast gels, Bio-Rad, cat. no. 456-1093) and blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was incubated overnight at 4°C with primary antibodies (1 : 3000) against RAF-1 and PDX-1 (Santa Cruz). Anti-β-actin (Santa Cruz, sc-7210) antibodies were used to ensure the quality of protein separation and loading contents. After extensive washes, the membranes were incubated with HRP-conjugated IgG secondary antibodies (Santa Cruz, sc-2004) and visualized with enhanced chemiluminescence (Amersham Life Sciences, UK) using a gel imaging system (Biospectrum 410, UVP).
Pdx 1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
PDX-1 is a laboratory reagent used in scientific research. It functions as a transcription factor involved in the regulation of insulin gene expression. The core function of PDX-1 is to serve as a research tool for studying pancreatic development and insulin homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Pgc 1α, by Santa Cruz Biotechnology (2 mentions)
Foxo1, by Cell Signaling Technology (2 mentions)
Hrp conjugated igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Biospectrum 410, by Analytik Jena (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Catalase, by Santa Cruz Biotechnology (1 mentions)
Antibodies against caspase 3, by Santa Cruz Biotechnology (1 mentions)
Acetylated lysine, by Cell Signaling Technology (1 mentions)
Dynabeads protein a immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Total histone h3, by Cell Signaling Technology (1 mentions)
Hdac4, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P foxo1 ser256, by Cell Signaling Technology (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Collagenase p, by Roche (1 mentions)
7 protocols using pdx 1
1
Western Blot Analysis of RAF-1 and PDX-1
2
Molecular Mechanisms in Cell Biology
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All common chemicals, including methylthiazol-2-yl-3 (4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO), propidium iodine (PI), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and JC-1, were purchased from Sigma (München, Germany). Antibodies against caspase 3, poly(ADP-ribose) polymerase (PARP), PDX-1, MST1, catalase, Nrf2, and PGC-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β-actin was from Novus Biotechnology (Littleton, CO, USA); RANK was from Cell Signaling Technology (Danvers, MA, USA); pSer536-p65 was from Abcam (Cambridge, MA, USA); and SOD1, SOD2, and Sirt1 antibodies were from GeneTex (Irvine, CA, USA). Denosumab was purchased from GlaxoSmithKline (London, UK). Lentiviruses carrying MST1, RANK, and Sirt1 specific short hairpin (sh)RNAs were purchased from the Taiwan National RNAi Core Facility. All chemicals were dissolved in phosphate-buffered salt (PBS) solution and stored at 20 °C until use in experiments.
3
Immunoblotting and Immunoprecipitation Assay Protocol
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Immunoblotting and immunofluorescence staining were carried out as previously described (Jing et al., 2011 ). Immunoprecipitation was performed using Dynabeads Protein A Immunoprecipitation Kit according to the manufacturer's protocol (#10006D, Thermofisher, USA). Antibodies used in this study: HDAC4 (#7628, Cell signaling, Germany), FoxO1 (#2880, Cell signaling, Germany), Pdx1 (sc‐14664, Santa Cruz, USA), acetylated lysine (#9441, Cell signaling Germany), α‐Tubulin (#T6199, Sigma, USA), and total histone H3 (#4499, Cell signaling, Germany) (Table S4 ).
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
4
Evaluating FLZ Compound Effects
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FLZ, a white powder with 99% purity, was kindly provided by Professor Dan Zhang from Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342 (HO), propidium iodide (PI), sodium pyruvate, glutamine, and beta-mercaptoethanol were purchased from Sigma (St. Louis, MO, USA). MK-2206 was purchased from Selleck Chemicals (Houston, TX, USA). Collagenase P was provided by Roche Molecular Biochemicals (Indianapolis, IN, USA). PDX-1, Akt, Lamin B, and β-actin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). p-Akt (Ser473), p-FOXO1 (Ser256), and FOXO1 antibodies were obtained from Cell Signaling (Danvers, MA, USA). Goat and rabbit secondary antibodies were product of Sigma (St. Louis, MO, USA). Rat and mouse Insulin Ultrasensitive ELISA kit were purchased from ALPCO Diagnostics (Salem, NH, USA). Nuclear-cytosol extraction kit was purchased from Applygen Technologies (Beijing, China). ADP/ATP assay kit was provided by BioVision (Mountain View, CA, USA).
5
Western Blot Analysis of Cell Proteins
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Lysates of cells or tissues were prepared with RIPA buffer (Beyotime Biotechnology, China). Equal amounts (20-30 μg) were subjected to SDS‒PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane filter. After incubation with the appropriate antibodies against ZHX2 (Proteintech), PCNA (Abcam), PAX6 (Santa Cruz Biotechnology), MAFA (Santa Cruz Biotechnology), PDX1 (Santa Cruz Biotechnology), insulin (Santa Cruz Biotechnology), GLUT2 (Santa Cruz Biotechnology), HA (MBL), β-actin (Proteintech), and GAPDH (Proteintech), secondary antibodies against rabbit IgG (Proteintech) and mouse IgG (Proteintech) were incubated.
6
Investigating Oxidative Stress and Apoptosis
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We purchased reagents, such as palmitic acid (PA), oleic acid (OA), 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), 4′,6‐diamidino‐2‐phenylindole (DAPI), acridine orange (AO), dihydroethidium (DHE) and 2′,7′‐dichlorodihydrofluorescein diacetate (H2‐DCFDA), from Sigma‐Aldrich (München, Germany). Mst1 siRNA (#AM16708) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). We purchased antibodies against caspase 3, poly(ADP‐ribose) polymerase (PARP), PDX1, Mst1, Lamin B1, p‐PERK, p‐eIF2α, AMPK, p‐AMPK, Nrf2 and PGC1α from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against LC3 and β‐actin were obtained from Novus Biologicals (Littleton, CO, USA), and antibodies against SOD1 and Sirt1 were purchased from GeneTex (Irvine, CA, USA). Liraglutide was purchased from Novo Nordisk (Copenhagen, Denmark). All chemicals were dissolved in phosphate buffer saline (PBS) solution and stored at −20°C until used in the experiments.
7
Immunofluorescence Staining of Stem Cells
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Cells were washed with 1! PBS for three times and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by 10 min permeabilization with 0.2% Triton X-100 in PBS. The cells were blocked for 1 h with 5% goat serum and 2% BSA. Diluted primary antibodies were then added to the culture and incubated at 4 8C overnight. IgG isotype control mouse and rabbit polyclonal IgG (Abcam, Cambridge, MA, USA) were used as control for staining respectively. After four washes with 1! PBS, diluted secondary antibodies against specific primary antibodies were added and incubated in the dark at room temperature for 1 h. 4 0 ,6-Diamidino-2-phenylindole (DAPI, Invitrogen) was added 10 min before the final wash. Pictures were taken under fluorescent microscope. The primary antibodies used were OCT4 (mouse, 1:200 dilution, Santa Cruz), SOX9 (rabbit, 1:300 dilution, Santa Cruz), PDX1 (rabbit, 1:300 dilution, Santa Cruz), and FOXA2 (mouse, 1:200 dilution, Santa Cruz). The secondary antibodies used were Alex 488 (goat anti-mouse, 1:500, Life Technologies) and Alexa 568 (goat anti-rabbit, 1:500, Life Technologies).
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