At 48 h following transfection with siRNA and recombinant ID1 plasmids, total proteins were extracted from SACC-83 cells with IP lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The protein concentration was determined by BCA method and 25 µg of protein was loaded per lane for SDS-PAGE separation. The proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (GE Healthcare; Chicago, IL, USA). Membranes were blocked in 5% bovine serum albumin (VWR International, Radnor, PA, USA) at 4°C for 60 min. Subsequently, the membranes were immunoblotted overnight at 4°C with primary antibodies against ID1 (ab66495; 1:1,000; Abcam) or GAPDH (M20006; 1:2,000; Abmart, Inc., Berkeley Heights, NJ, USA). Membranes were then washed three times in Tris-buffered saline with 0.1% Tween 20 and subsequently incubated with a secondary antibody (Goat Anti-Mouse IgG H&L (HRP); ab205719; 1:2,000; Abcam) at room temperature for 60 min. Protein bands were visualized using CDP-Star reagent (Roche Diagnostics, Indianapolis, IN, USA). The signals were detected by exposure to X-Ray film for different times (1–10 min).
M20006
Manufactured by Abmart
Sourced in United States
The M20006 is a laboratory equipment product. It is designed to perform a core function in a laboratory setting. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Cdp star reagent, by Roche (1 mentions)
Ip lysis buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Ab66495, by Abcam (1 mentions)
Goat anti mouse igg h l hrp ab205719, by Abcam (1 mentions)
Ab7766, by Abcam (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab2868, by Abcam (1 mentions)
A5865, by ABclonal (1 mentions)
Ab85476, by Abcam (1 mentions)
A19544, by ABclonal (1 mentions)
Ab1424, by Abcam (1 mentions)
M20008, by Abmart (1 mentions)
Ab8227, by Abcam (1 mentions)
5 protocols using m20006
1
Western Blot Analysis of ID1 Protein
2
UHRF1 Protein Domain Plasmid Construction
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The plasmids for pGEX4T-1-UHRF1 (aa 95–610) and pGEX4T-1-UHRF1 (aa 1–407) was as described52 (link). The pGEX4T-1-UHRF1 (aa 95–610)-YP191/192AA was derived from pGEX4T-1-UHRF1 (aa 95–610) by site-directed mutagenesis. Antibodies used in this study were as follows: anti-UHRF1 as described52 (link), anti-H3 (Abcam, #ab7766), anti-AcH3 (Abcam, #ab47915), anti-H3K27me3 (CST, #9733S), anti-H3K9me3 (Abclonal, #A2360), anti-GST (HuaAn, #EM80701) and anti-GAPDH (AbMart, #M20006).
3
Immunohistochemical Analysis of Trait Genes
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HCC tissues were paraffin-embedded, sectioned, dewaxed and hydrated, incubated with anti-trait gene antibodies overnight at room temperature, then labeled with secondary antibodies for 30 min, stained and photographed.
The specific primary antibodies were purchased from the following resource:
GAPDH(Abmart, M20006, WB(1:5000)), CBX2(Abmart, PH3521, WB(1:1000), IHC(1:100)), CDKN2B(Affbiotech, AF0230, WB(1:1000), IHC(1:100)), ETS2(Abmart, MG225391, WB(1:1000), IHC(1:150)), HMGA1(ABclonal, A4343, WB(1:1000), IHC(1:100)), UBE2S(Abmart, PK66136, WB(1:1000), IHC(1:100)).
The specific primary antibodies were purchased from the following resource:
GAPDH(Abmart, M20006, WB(1:5000)), CBX2(Abmart, PH3521, WB(1:1000), IHC(1:100)), CDKN2B(Affbiotech, AF0230, WB(1:1000), IHC(1:100)), ETS2(Abmart, MG225391, WB(1:1000), IHC(1:150)), HMGA1(ABclonal, A4343, WB(1:1000), IHC(1:100)), UBE2S(Abmart, PK66136, WB(1:1000), IHC(1:100)).
4
Protein Expression Analysis of iPSC-Derived Cardiomyocytes
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The iPSC-CMs in a well of 6-well plate were detached with Trypsin–EDTA (Gibco, 25200056), and then pelleted at 300 g for 3–5 min at 4 °C. After washing with DPBS, the pellets were re-suspended in 50–100 μl lysis buffer. Lysates were placed on ice for 30 min and the supernatants were collected after centrifuging at 12,000 rpm for 5 min. Protein concentration was measured using a BCA kit (Pierce, 23227). Western blot was performed with the following antibodies: SERCA2a (Santa Cruz Biotechnology, sc-53010, 1:200, RRID: AB_630230 ), RYR2 (abcam, ab2868, 1:500, RRID: AB_2183051 ), sodium/calcium exchanger 1 (NCX1) (ProteinTech, 55075-1-AP, 1:500, RRID: AB_2881262 ), Cav 1.2 (ProteinTech, 21774-1-AP, 1:2000, RRID: 21774-1-AP), total phospholamban (PLN) (Cell Signaling Technology, 14562S, 1:1000, RRID: AB_2798511 ), phosphorylated PLN (Cell Signaling Technology, 8496S, 1:1000, RRID: AB_10949102 ), Nav1.5 (Alomone Labs, ASC-005, 1:500, RRID: AB_2040001 ), and GAPDH (Abmart, M20006, 1:5000, RRID: AB_2737054 ). Intensity values for each band were determined as the integrated density (sum of pixel values) within a fixed area using Quantity One software (Biorad).
5
Western Blot Analysis of Iron Regulators
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Western blot analysis was performed as previously described [24 (link)]. Antibodies in this work were anti-KLF14 (ab85476; Abcam, Cambridge, MA, USA), anti-IRP1 (12406–1-AP, Proteintech, 1:1000), anti-IRP2 (23829–1-AP, Proteintech, 1:1000), anti-TfR1 (A5865, ABclonal, 1:1000), anti-Ferritin H (A19544, ABclonal, 1:1000), anti-SIRT1 (A17307, ABclonal, 1:1000), anti-GAPDH (M20006, Abmart, 1:5000), anti-Flag (M20008, Abmart, 1:5000, 1:1000), anti-HA (ab1424，abcam, 1:1000), anti-Actin (ab8227, abcam, 1:1000).
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