The whole-cell lysate of Hela and SiHa cells was extracted using lysis buffer (0.05 M Tris, pH 7.5, 0.15 M NaCl, 2% NP-40) containing 200 mM of Na3VO4, 200 mM of NaF, 0.5 M of EDTA, and proteinase inhibitors for 30 min on ice and then quantified by BCA protein assay kit. Proteins (20 µg) were separated by SDS-PAGE and then were transferred onto a PVDF membrane. The following antibodies were used for Western blot: GAPDH (10494-1-AP; Proteintech) and HOXA1 (13513-1-AP; Proteintech).
Gapdh 10494 1 ap
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
GAPDH (10494-1-AP) is a primary antibody product offered by Proteintech. It targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, which is a key enzyme involved in the glycolytic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Bcl 2 12789 1 ap, by Proteintech (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Tnf α, by Shanghai Enzyme-linked (3 mentions)
Il 1β, by Shanghai Enzyme-linked (3 mentions)
Il 10, by Shanghai Enzyme-linked (3 mentions)
Ab207305, by Abcam (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Lc3a b, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Zb 2301, by ZSGB-BIO (2 mentions)
Pd l1 13684s, by Cell Signaling Technology (2 mentions)
P27 25614 1 ap, by Proteintech (2 mentions)
Cat 21260 1 ap, by Proteintech (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ecl plus kit, by Beyotime (2 mentions)
β actin 20536 1 ap, by Proteintech (2 mentions)
72 protocols using gapdh 10494 1 ap
1
HeLa and SiHa Cell Lysate Extraction
2
Autophagy Modulation via Pharmacological Inhibitors
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SP600125 (HY-12041), wortmannin (HY-10197), rapamycin (HY-10219), chloroquine (CHQ) (HY-17589A), MG132 (HY-13259), and Z-VAD-FMK (HY-16658B) were purchased from MedChemExpres (Monmouth Junction, NJ, USA). Cycloheximide (CHX) (S7418) was purchased from Selleck (Shanghai, China). Psi-U6.1/eGFP/Puro-FUT2 and psi-U6.1/eGFP/Puro-NC vectors were designed by GeneCopoeia (Guangzhou, China). StubRFP-sensGFP-LC3 Lentivirus and FUT2 cDNA expression and control vectors were constructed by Shanghai Genechem. SiDRAM1 (C6133) was purchased from Shanghai GenePharma. Antibodies for LC3-II (18725-1-AP), Apaf-1 (21710-1-AP), Bcl-2 (12789-1-AP), cleaved Caspase-9 (10380-1-AP), p53 (10442-1-AP), and GAPDH (10494-1-AP) were purchased from Proteintech (Chicago, IL, USA). Antibodies specific against FUT2 (sc-100742), p-JNKTyr185, Thr183 (81E11), JNK (sc-7345) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for AMPK (db1167), Cytc (db2293), ULK1 (db2872), Tubulin (db3285), and β-actin (db10001) were purchased from Diagbio (Hangzhou, China). p-PI3K III (#13857) was purchased from Cell Signaling Technology (Danvers, MA, USA). Others antibodies for Beclin1 (ab207612), p62 (ab207305), p-AMPKThr183, Thr172 (ab133448), p-ULK1 (ab133747), Atg5 (ab108327), DRAM1 (ab64739), and pro Caspase-9 (ab32539) were purchased from Abcam (Boston, MA, USA).
3
Antibody Characterization and TGF-β1 Signaling
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Antibodies were purchased from the following companies: Smad‐2/3(D7G7), α‐SMA (#19245), vimentin (#5741) and β‐actin (#P60709) were purchased from Cell Signalling Technology; TGF‐β1 (ab215715) was purchased from Abcam; NF‐κB (10745‐1‐AP) and GAPDH (10494‐1‐AP) were obtained from Pro‐teintech; CHRFAM7A (860647) was purchased from ZEN BIO; Kidney injury molecule 1(KIM‐1) (KCA0319031) antibodies was purchased from R&D system. Recombinant human TGF‐β1 were obtained from Pepro Tech (121809). Lipofectamine 2000 was purchased from Thermo Fisher (11668‐019) and the reverse transcription cDNA synthesis system and SYBR Green were obtained from TIANGEN.
4
Western Blot and Co-Immunoprecipitation Assay
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The following antibodies were used for western blotting: p53 (OP43; Merck Millipore), MDM2 (SMP14) (sc-965; Santa Cruz), AKT (2920; Cell Signaling Technology), phospho-AKT (Thr308) (9275; Cell Signaling Technology), phospho-AKT (Ser473) (9271; Cell Signaling Technology), PARP (9532; Cell Signaling Technology), caspase 3 (9661; Cell Signaling Technology), phospho-5′ AMP-activated protein kinase ([AMPK] bs-4002R; Bioss), AMPK (E-AB-30490; Elabscience Biotechnology Inc.), phospho-mammalian target of rapamycin ([mTOR] (E-AB-20929; Elabscience Biotechnology Inc.), mTOR (E-AB-32129; Elabscience Biotechnology Inc.), LC3A/B (4108; Cell Signaling Technology), α-tubulin (NB100-690; Novus Biologicals), GAPDH (10494-1-AP; Proteintech), HSP-40 (sc-59554; Santa Cruz Biotechnology), CHIP (sc-133066; Santa Cruz Biotechnology), and vinculin (GTX109749; GeneTex). For co-immunoprecipitation assay, cell lysates were incubated with p53 antibody (OP43, EMD Millipore, Billerica, MA) or HSP-40 antibody (sc-398766, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Then samples were precipitated with appropriate protein A/G beads with matched IgG as negative control. The precipitants were analysed in SDS-PAGE western blot.
5
Cellular Protein Extraction and Western Blot
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Cellular proteins were extracted from PC-3 and DU145 cells with NF and CAF supernatant, and protein concentrations were determined using the BCA kit (P0011, Beyotime, China). Western blot was performed as described [23 (link)]. The antibodies used in this study include FAP (66,562 S, CST, USA), PDGFRα/β (ab5443, Abcam, UK), α-SMA (A17910, ABclonal, USA) and GAPDH (10494-1-AP, Proteintech, USA).
6
Immunoblot Analysis of Let-7c Inhibition
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The cells were transfected with a Let-7c inhibitor. Additionally, 1X SDS lysis buffer was used to lyse the cells. SDS-PAGE was used to isolate proteins, which were transferred onto PVDF membranes. MYC (#9402, Cell Signaling Technology, Massachusetts, USA) and GAPDH (10,494–1-AP, Proteintech, Wuhan, Hubei, China) staining was detected. The Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln Nebraska, USA) was used to visualize immunoreactive bands.
7
Rapamycin Modulates Fibrosis and Autophagy
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Adenine (A8330) was obtained from Solarbio Life Science (Beijing, China). Recombinant human TGF-β1 protein (10804-HNAC) was purchased from SinoBiological (Beijing, China). Rapamycin (HY-10219) was obtained from MedChemExpress Biotech Co., Ltd. (Monmouth Junction, NJ, USA). Antibodies of fibronectin (ab268020), α-SMA (ab7817), ALDH2 (ab108306), and Ki67 (ab16667) were bought from Abcam (Cambridge, MA, USA). p-ULK1ser757 (14202s), p-AKT (4060s), SQSTM1 (5114s), and AKT (4691s) were from Cell Signaling Technology (Danvers, MA, USA). Vimentin (60330-1-Ig), p53 (60283-2-Ig), p-H3ser10 (66863-1-Ig), p-mTOR (67778-1-Ig), mTOR (66888-1-Ig), ULK1 (20986-1-AP), Lc3 (14600-1-AP), and GAPDH (10494-1-AP) were from Proteintech (Chicago, IL, USA). ATG7 (ET1610-53) and Beclin-1 (R1509-1) were from HuaBio (Hangzhou, China). E-cadherin (A20798) was from ABclone Technology (Wuhan, China). p-p53ser15 (530083) was from Zen-Bioscience (Chengdu, China). p21 (A5163) was brought from Bimake (Houston, TX, USA). Anti-α-tubulin (M03989-2) was from Boster (Wuhan, China). All other reagents and chemicals, unless indicated, were obtained from Sangon Biotech (Shanghai, China).
8
Hinokiflavone Inhibits Cancer Cell Proliferation
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Hinokiflavone (HF) with purity > 97% was purchased from Chengdu Herbpurify CO., LTD (Chengdu, China). SP600125, SB203580, U0126, 3-Methyladenine (3-MA) and Chloroquine phosphate (CQ) were obtained from MedChemExpress (New Jersey, USA). p-p38 (4511S), p38 (8690S), p-ERK1/2 (9101S), ERK1/2 (9102S), p-MEK1/2 (9154S), MEK1/2 (9122S), p-JNK (9251S), JNK (9252S), p-NF-κB p65 (3033S), NF-κB p65 (8242S), Cyclin B1 (12231S), Cdc2 (9116S), p21 (2947S), Cleaved caspase-3 (9664S), Cleaved caspase-9 (7237S), Cleaved PARP (9546S), and LC3 (12741S) were provided by Cell Signaling Technology (Beverly, MA, USA). p62 (ab82645) was provided by Abcam (Cambridge, UK). GAPDH (10494–1-AP) was provided by Proteintech Group (Wuhan, China). Anti-rabbit (A0208) and anti-mouse (A0216) secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China).
9
Molecular Mechanism of STAT3 Regulation
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Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-STAT3 (Tyr705) (#9145, 1:1000), STAT3 (#30835, 1:2000), p-p65(Ser536) (#3033, 1:1000), p65 (#8242, 1:2000), and α-SMA (#19245, 1:2000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-1β (sc-12742, 1:2000), CD34 (sc-7324, 1:2000), VCAM-1 (sc-13160, 1:2000), and EPAS-1 (sc-13596, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD68 (28058-1-AP), ICAM-1 (60299-1-Ig, 1:2000), MCP-1 (66272-1-Ig, 1:2000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:2000), cleaved-caspase-3 (19677-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000), FN1 (15613-1-AP, 1:2000), and SOX9 (67439-1-Ig, 1:2000) as well as secondary antibodies (Goat anti-mouse, SA00001-1; Goat anti-rabbit, SA00001-2) were purchased from Proteintech Group (Chicago, IL, USA). Recombinant human interleukin-1 receptor antagonist (IL-1Ra) (SRP3084) was obtained from Sigma (St. Louis, MO, USA), Stattic (inhibitor of STAT3) and Colivelin, (activator of STAT3) were purchased from MedChemExpress (Shanghai, China). Unless otherwise indicated, the remaining reagents used in this study were obtained from Sigma.
10
Molecular Mechanisms of DHA and β-Estradiol
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DHA (D2534) and β-estradiol (E8875) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies of β-catenin (66379-1-Ig), GSK3β (22104-1-AP), and GAPDH (10494-1-AP) were obtained from Proteintech (Chicago, IL, USA). Antibodies against phospho-β-catenin (DF2989) and phospho-JNK (AF3318) were from Affinity (Ancaster, ON, Canada). Antibodies for JNK (9252) and phospho-GSK3β (D3A4) were purchased from Cell Signaling Technology (Danvers, MA, USA).
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