After homogenizing the tissues and cells using lysis buffer and centrifugation at 12,000 g for 20 min at 4 °C, protein amounts from all samples were measured with the BCA-kit (Thermo Fisher Scientific; Waltham, MA, USA). Protein samples (50 μg) were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto an immobilon-FL transfer membrane (Millipore, Billerica, MA, USA) in a transferring buffer. The membrane was blocked with 5% milk in tris-buffered saline tween-20 (TBST) for 1 h and then incubated overnight at 4 °C with antibodies against TGF-β1, p-smad2, smad2, p-smad3, smad3, smad4, and C-C chemokine receptor (CCR) 2, which were purchased from Cell Signaling Technology (Boston, MA, USA). GAPDH (MB001) was purchased from Bioworld Technology (St Louis Park, MN, USA). The blots were scanned using a two-color infrared imaging system (LI-COR Biosciences: Lincoln, NE, USA). Specific protein expression levels were normalized to GAPDH protein for total cell lysates.
Gapdh mb001
Manufactured by Bioworld Technology
Sourced in United States
GAPDH (MB001) is a lab equipment product that serves as an important enzyme involved in the glycolytic pathway. It catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, an essential step in cellular energy production.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Hrp conjugated goat anti rabbit igg, by EarthOx (2 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Immobilon fl transfer membrane, by Merck Group (1 mentions)
Antibodies against tgf β1, by Cell Signaling Technology (1 mentions)
Two color infrared imaging system, by LI COR (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Apigenin, by Merck Group (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Naringenin, by Merck Group (1 mentions)
Hesperetin, by Merck Group (1 mentions)
Antibody against lamin b, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Chenodeoxycholic acid, by Merck Group (1 mentions)
Ab76541, by Abcam (1 mentions)
Cadherin 17, by Cell Signaling Technology (1 mentions)
Ab128908, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Foxo4, by Cell Signaling Technology (1 mentions)
7 protocols using gapdh mb001
1
Western Blot Analysis of TGF-β1 Signaling
2
Antiviral Compounds and Antibodies
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Flavonoids including apigenin, kaempferol, hesperetin, naringenin, and other chemicals used for antiviral studies were purchased from Sigma-Aldrich (St. Luis, MO). Rabbit anti-EV71 VP1 was purchased from Abnova (Taiwan). A mouse antiserum to EV71 was prepared in the lab by immunizing mice with UV-deactivated EV71 and used for immunostaining studies. Antibodies against human hnRNP A1 (BS2255), hnRNP A2/B1 (BS6196), and GAPDH (MB001) were obtained from Bioworld Technology (Minneapolis, MN). Antibody against Lamin B was obtained from Santa Cruz Biotechnology (Dallas, TX). HRP-conjugated secondary antibodies were purchased from Bio-Rad, and Alexa Fluor-conjugated antibodies were from Life Technologies (Carlsbad, CA).
3
Molecular Mechanisms of Cell Signaling
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Chemical reagents (obtained from Med‐Chem Express, MCE, USA) were dissolved in dimethyl sulfoxide (DMSO; Lot #SHBH2446V, Sigma‐Aldrich, USA) for storage in a −80°C freezer chenodeoxycholic acid (CDCA; Cat. No. HY‐76847), 200 μM; resveratrol (Res; Cat. No. HY‐16561), 200 μM; LY294002 (Cat. No. HY‐10108), 50 μM.
Anti‐β‐actin (A1978, 1:5000) was purchased from Sigma‐Aldrich (USA), and GAPDH (MB001, 1:5000) and tubulin β (BS1482M, 1:5000) were purchased from Bioworld Technology (USA). Primary antibodies against human CDX2 (ab76541, 1:1000), and FoxO4 (ab128908, 1:1000), FoxO4 (phospho‐Ser262, ab126594, 1:10000) were from Abcam (UK). Primary antibodies against human CDX2 (#12306, 1:1000), Klf4 (#12173, 1:1000), Villin‐1 (#2369, 1:1000), cadherin‐17 (#42919, 1:1000), FoxO4 (#9472, 1:1000), AKT (#9272, 1:1000), phospho‐AKT Ser473 (#9271, 1:1000) and phospho‐AKT Thr308 (#9275, 1:1000) were purchased from Cell Signalling Technology (USA).
Anti‐β‐actin (A1978, 1:5000) was purchased from Sigma‐Aldrich (USA), and GAPDH (MB001, 1:5000) and tubulin β (BS1482M, 1:5000) were purchased from Bioworld Technology (USA). Primary antibodies against human CDX2 (ab76541, 1:1000), and FoxO4 (ab128908, 1:1000), FoxO4 (phospho‐Ser262, ab126594, 1:10000) were from Abcam (UK). Primary antibodies against human CDX2 (#12306, 1:1000), Klf4 (#12173, 1:1000), Villin‐1 (#2369, 1:1000), cadherin‐17 (#42919, 1:1000), FoxO4 (#9472, 1:1000), AKT (#9272, 1:1000), phospho‐AKT Ser473 (#9271, 1:1000) and phospho‐AKT Thr308 (#9275, 1:1000) were purchased from Cell Signalling Technology (USA).
4
Western Blot Analysis of Cellular Proteins
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Total cell lysates were prepared by suspending cells in the RIPA buffer (Beyotime, China) supplemented with 1× complete protease inhibitors mixture and 1× phosphatase inhibitor (Roche). Protein concentration was determined by BCA assay (Pierce, Rockford, USA). Equal quantities of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and blotted with specific antibodies. Protein in the membrane was visualized by an enhanced chemiluminescence detection kit (Millipore, USA). Antibodies against the following proteins were used, with catalog numbers and sources listed: CD317 (1C12) [18 (link)] (in-house); GAPDH (MB001) (Bioworld Technology); β-actin (A5441) (Sigma); K48-Linkage-Specific Ubiquitin (ab140601) (Abcam); Calnexin (00050122) (Proteintech); CHOP (L63F7) (CST); β-tubulin (200608) (ZEN BIO); HRP-conjugated mouse anti-HA1.1 tag (901519) (Biolegend); HRP-conjugated mouse anti-Flag (200-303-383) from Rockland; HRP-conjugated goat anti-mouse IgG (074-1806) from KPL, and HRP-conjugated goat anti-rabbit IgG (E030120-02) from EARTHOX. Full and uncropped western blots are presented in Supplemental File (Supplementary Fig. S7) .
5
Protein Expression Analysis Protocol
6
Quantifying AKT1 Regulation by miRNAs
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For western analysis of AKT1, cells were plated in 2i/Lif media 24 h before transfection of 50 nM miR-294 or miR-302a. Proteins were extracted 32 h after transfection. Similar results were obtained for proteins extracted 46 h after transfection. Antibodies against ERK (#9102), pERK (#9101) and AKT1 (#2938) were from Cell Signaling Technology (Massachusetts, USA), against GAPDH (MB001) was from Bioworld Technology (Nanjing, China).
7
Western Blot Analysis of ERK Phosphorylation
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Antibodies against ERK (#9102), pERK (#9101) were from Cell Signaling Technology, against GAPDH (MB001) was from Bioworld Technology (Nanjing, China). Against FLAG (F1804) was from Sigma. Anti-rabbit (926–32213) and mouse (926–68022) secondary antibodies were from LI-COR Biosciences and membranes were imaged using Oddssey. For background pERK analysis in 2i + LIF and PD + LIF condition (Figs. 2 d, 2f and 6b ), because the signal was too weak, HRP-conjugated anti-rabbit secondary antibodies were used and membranes were incubated with the Western ECL Substrate (WBKLS0500, Milipore) and imaged using Amersham imager 600 (GE Life Sciences). All primary antibodies were used at a dilution of 1:1000. All secondary antibodies were used at a dilution of 1:10000. For the analysis of pERK induced by bFGF, cells were plated in 2i + LIF for ~24 h, then grown in CHIR (to remove the interfering effects of PD and LIF on ERK phosphorylation) for ~48 h before bFGF induction, proteins were extracted 15 min after the addition of 12 ng per ml bFGF. Unprocessed images for all western blots in main figures are shown in Supplementary Figure 8 .
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