Cellular protein samples were extracted using RIPA lysis buffer. After quantification with the BCA Protein Assay Kit (Beyotime, China), the concentration of each sample was adjusted. Samples were mixed with SDS-PAGE protein loading buffer and stored at −20°C. The mixed samples were electrophoresed with 120V for 1h and transferred to a 0.45-um poly vinylidene fluoride membrane. Then, the membrane was blocked and incubated with primary antibody for 12h (4°C), followed by secondary antibody incubation for 1h and detection (ChemiDoc MP; Bio-Rad, USA). The antibodies used in this study were β-actin rabbit monoclonal antibody (AF5003, Beyotime, China), CDK1 rabbit polyclonal antibody (AF0111, Beyotime, China), Phospho-CDK1 (pCDK1, Tyr15) rabbit polyclonal antibody (AF5761, Beyotime, China), and HRP-labeled goat anti-rabbit IgG (H+L) (A0208, Beyotime, China).
Hrp labeled goat anti rabbit igg h l
Manufactured by Beyotime
Sourced in China, United States
HRP-labeled goat anti-rabbit IgG (H+L) is a secondary antibody that binds to rabbit immunoglobulin G (IgG) molecules, including both the heavy (H) and light (L) chains. The antibody is conjugated with horseradish peroxidase (HRP), which can be used as a detection label in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hrp labeled goat anti mouse igg h l, by Beyotime (25 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Bca protein assay kit, by Beyotime (9 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Ecl reagent, by Beyotime (4 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Slicing machine, by Leica (2 mentions)
Tris edta buffer ph 9, by Solarbio (2 mentions)
Nbp2 32356, by Novus Biologicals (2 mentions)
Pannoramic midi, by 3DHISTECH (2 mentions)
Caseviewer 2, by 3DHISTECH (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Antifade mounting medium, by Beyotime (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (2 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (2 mentions)
69 protocols using hrp labeled goat anti rabbit igg h l
1
Western Blot Analysis of Cell Proteins
2
HOXA9 Expression Analysis in Muscle Tissue
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Mid-portions of the operational gastrocnemii were xed through paraformaldehyde (4%), then dehydrated, and para n-embedded. 5-μm thick cross-sectional slices of the muscle wax samples were made on a slicing machine (Leica Biosystems, Germany). Following depara nage, hydration, heat mediated antigen retrieval (Tris/EDTA buffer PH 9.0, Solarbio), endogenous peroxidase activity blocking, and goat serum blocking, primary rabbit anti-HOXA9 (1:500; NBP2-32356, Novus, US) incubation was conducted overnight at 4 °C. After application of HRP-labeled Goat Anti-Rabbit IgG(H+L) (1:50, Beyotime, Haimen, China), diaminobenzidine working solution (DAB Horseradish Peroxidase Color Development Kit, Beyotime), the sections were counter-stained with hematoxylin. Images were scanned and analyzed through Pannoramic MIDI (3DHISTECH Ltd, Hungary) and Caseviewer 2.2 (3DHISTECH Ltd, Hungary).
3
Western Blot Analysis of Protein Targets
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Cells were lysed with RIPA Lysis Buffer (P0013B, Beyotime, China) or Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, China). Then protein concentrations were quantified with BCA Protein Assay Kit (P0011, Beyotime, China). Twenty microlitre of proteins were separated by SDS-PAGE and then transferred onto PVDF membranes. After blocking, the membranes were incubated with the one of following antibodies: anti-CARMA3 antibody (1:1000, bs-7081R, Bioss, China), anti-MMP2 antibody (bs-0412R, Bioss), anti-MMP9 antibody (1:1000, bs-4593R, Bioss, China), anti-β-catenin antibody (1:1000, #8480, Cell Signaling Technology, USA), anti-Survivin antibody (1:1000, #2808, Cell Signaling Technology, USA), anti-C-myc antibody (1:500, sc-40, Santa Cruz Biotechnology, USA), anti-beta-Actin (1:5000, bsm-33036 M, Bioss, China), or anti-Histone H3.1 (1:500, bs-17422R, Bioss, China) at 4 °C overnight. Then the membranes were incubated in HRP-labeled Goat Anti-Rabbit IgG (H + L) (1:5000, A0208, Beyotime, China) or HRP-labeled Goat Anti-Mouse IgG (H + L) (1:5000, A0216, Beyotime, China) at 37 °C for 45 mins. After incubating ECL reagent for 5 mins, the proteins were detected by ECL Select Western Blotting Detection System (Beyotime, China).
4
Selenium-Enriched Soybean Antioxidant Analysis
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Se-enriched soybeans were acquired from Enshi Se-Run Health Tech Development Co., Ltd. (Enshi, China). The PM2.5 standard reference material (SRM 2786) [26 ] was procured from the NIST (Gaithersburg, MD, USA). Seleno-DL-methionine (SeMet) and sodium selenite (Na2SeO3) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). DMEM, FBS, penicillin, and streptomycin were acquired from Gibco (Grand Island, NE, USA). The ELISA kits for IL-1β, IL-6, and TNF-α were purchased from Multisciences (Hangzhou, China). The antibodies against Bax (AF1270), Bcl-2 (AF6285), caspase-3 (AF1213), Akt (AF1777), p-Akt (Ser473) (AA329), β-Actin (AF5003), and HRP-labeled goat anti-rabbit IgG (H + L) were obtained from Beyotime (Shanghai, China). The Hoechst 33342, ECL kit, CCK-8 kit, and ROS kit were supplied by Beyotime (Shanghai, China). The RIPA lysis buffer, Annexin V-FITC/PI apoptosis kit, and BCA protein assay kit were supplied by Solarbio (Beijing, China).
5
Protein Extraction and Western Blot Analysis
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Protein was extracted from CD4+ T cells using the Minute Total Protein Extraction Kit for Animal Cultured Cells (SD-001, Invent Biotechnologies, MN, USA). The buffer contains a 1% mixture of protease and phosphatase inhibitors. The proteins were transferred to PVDF membranes by electrophoresis using SDS-PAGE gels (Epizyme, Shanghai, China). Blocking treatment was performed using NcmBlot Blocking Buffer. Antibodies were against ASC/TMS1 (Proteintech, 10,500- 1-AP), NLRP3 (Proteintech, 19,771–1-AP), GLI1 (Proteintech, 66,905–1-Ig), Caspase-1 (ab207802) and ERAP2 (Abcam, ab69037) from Abcam (Cambridge, USA), GSDMD (NBP2-33,422) from Novus (CO, USA), SHH (sc-166685) and SMO (Santa Cruz, sc-365112) from Santa Cruz (TX, USA). HRP-labeled goat anti-rabbit IgG (H + L) (Beyotime, A0208) and HRP-labeled goat anti-mouse IgG (H + L) (Beyotime, A0216) were used as secondary antibodies. Anti-beta-actin (Proteintech, 66,009–1-Ig) was used as the gel-loading control. The membranes were incubated with Abs, and then Western ECL (P10300) from NCM Biotech (Jiangsu China) was added for imaging with the Bio-Rad image system. The gray value of each band was measured using ImageJ.
6
Western Blot Antibody Panel
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Anti-SOD2 antibody produced in goat (1:1000; Sigma, USA), anti-β-tubulin mouse monoclonal antibody (1:1000; TransGen Biotech, China), rabbit anti-rat caspase-1 antibody (1:1000; Abcam, England), rabbit anti-rat caspase-3 antibody (1:1000; Abcam, England), rabbit anti-rat IL-1β antibody (1:1000; Abcam, England), rabbit anti-rat IL-18 antibody (1:1000; Abcam, England), anti-GAPDH mouse monoclonal antibody (1:1000; TransGen Biotech, China). Secondary antibody: HRP-labeled goat anti-rabbit IgG (H + L) (1:1000; Beyotime, China), goat anti-mouse IgG (H + L) secondary antibody, HRP (Invitrogen, USA), rabbit anti-goat IgG HRP-conjugated antibody (1:1000; Gentex, USA), Toxoplasma gondii SAG1 monoclonal antibody (1:20; Invitrogen, USA), goat anti-mouse IgG (H + L) cross-adsorbed ReadyProbes™ secondary antibody, and alexa fluor 594 (1:1000; Invitrogen, USA).
7
Western Blot Analysis of Stem Cell Markers
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Cells were treated with RIPA Lysis Buffer (Cat #P0013B, Beyotime, Beijing, China), followed by determining protein concentration using Bradford Protein Assay Kit (Cat #P0006, Beyotime). Then 30 μg protein was separated by 10% SDS-PAGE. The protein was transferred onto PVDF membranes (Bio-Rad). Blocking was carried out with 10% non-fat milk for 1.5 hours at 37°C. Then the membranes were washed with TBST for 15 minutes, 3 times, and incubated with the primary antibodies at 4°C overnight. The primary antibodies against ALDH1 (ab129815), Nanog (ab80892), ABCG2 (ab203397), MDR1 (ab3366), p-mTOR (ab84400), cleaved caspase-3 (ab32042), and cleaved PARP (ab32064) were purchased from Abcam. The primary antibodies against β-actin (Cat #60008-1-Ig), p-Akt (Cat #66444-1-Ig), Akt (Cat #10176-2-AP), caspase-3 (Cat #19677-1-AP), PARP (Cat #13371-1-AP), E-cadherin (Cat #20874-1-AP), and vimentin (Cat #10366-1-AP) were purchased from Proteintech. Then membranes were washed with TBST for 15 minutes, 3 times, followed by incubating with HRP-labeled goat anti-rabbit IgG(H+L) (Cat #A0208, Beyotime), or HRP-labeled goat anti-mouse IgG(H+L) (Cat #A0216, Beyotime). ECL Plus (Cat #PE0010, Solarbio) was used to detect chemiluminescent signals in Bio-Rad ChemiDoc™ Touch (CM002554, Bio-Rad).
8
Detecting Thymidine Kinase Expression
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The expression of thymidine kinase protein was detected by western blot. Cell lysates were prepared from HEK293 cells transfected with an empty vector control (pIRES2-AcGFP1-Nuc) or different TK constructs (attB35TK, attBrTK, attBrG4STK, attBrP2ATK, and wt-TK). At 48 h post-transfection, the cells were harvested and re-suspended in phosphate buffered saline (PBS). Total protein (10 μg) was loaded per lane in a 12% SDS-PAGE gel. The gel was transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA) and subjected to western blot analysis according to a standard protocol. A goat polyclonal antibody against HSV-1 thymidine kinase (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and HRP-labeled Donkey Anti-Goat IgG (H + L; 1:1000; Beyotime, Jiangsu, China) were used to detect TK products. A rabbit polyclonal antibody against GAPDH (1:1000, Sigma) and HRP-labeled goat anti-rabbit IgG (H + L; 1:1000; Beyotime) were used to detect GAPDH, which was used as an internal control.
9
Investigating JIB04 regulation of cell signaling
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Dulbecco's modified Eagle's medium (DMEM, 11965092) and fetal bovine serum (FBS, 11011-8611) were purchased from Gibco (Invitrogen). Streptomycin and penicillin (GNM15140) were purchased from Genome Biotechnology. JIB04 (25 μM final concentration; HY-13953) was purchased from MCE. Anti-β-actin (AF5003, 1:1000), Anti-H3 (AF0009, 1:1000), anti-N-cadherin (AF5237, 1:1000), anti-p-ErK (AF1891, 1:1000), and anti-ErK (AF1051, 1:1000) were purchased from Beyotime Biotechnology. Tri-methyl-histone H3 (Lys4) (H3K4me3) (9751, 1:1000) was purchased from Cell Signaling Technology. Anti-PLU1 (ab181089, 1:1000) was purchased from Abcam. The secondary antibodies, HRP-labeled Goat Anti-Rabbit IgG (H + L) (A0208, 1:1000) and HRP-labeled Goat Anti-Mouse IgG (H + L) (A0216, 1:1000), were obtained from Beyotime Biotechnology.
10
Emodin Regulates Neuroinflammation Pathways
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The following
materials and reagents were used in the study: Emodin (no.: A0044,
Chengdu Must Biotechnology Co., Ltd., China, purity≥98%); anti-MMP9
(no.: Ab38898, Abcam, U.K.); anti-NF-κB p65 (no.: #8242, CST,
USA); anti-IKKβ (no.: #2678, CST, USA); 2,3,5-triphenyl tetrazolium
chloride (no.: T8877-25G, Sigma, USA); hematoxylin and eosin (HE)
staining kit (no.:BP-DL017, SenBeiJia Biological Technology Co., Ltd.
China); HRP-labeled goat anti-rabbit IgG (H + L) (no.: A0208, Beyotime
Biotechnology, China); anti-MMP2 (no.: Ab181286), anti-occludin (no.:
Ab216327), anti-extracellular signal-regulated kinase [ERK1/2 (no.:
Ab184699)], and p-anti-ERK1/2 (no.: Ab214036)—Abcam, U.K.;
IL-6 antibody (no.: DF6087), HIF1A antibody (no./AF1009), claudin-5
(no./AF5216), and VEGFA antibody (no.: DF7470)—Affinity, China;
Alexa Fluor 555-labeled donkey anti-mouse IgG(H + L) (A0460), Triton
X-100 (no.: ST795), and antifade mounting medium (no.: P0126)—Beyotime
Biotechnology, China; neutral balsam (no.: G8590, Beijing Solarbio
Science & Technology Co., Ltd. China); RNA Easy Fast Tissue/Cell
Kit (no.: DP451), FastKing gDNA Dispelling RT SuperMix (no.: KR118),
and Talent qPCR PreMix (SYBR Green) (no.: FP209)—Tiangen Biotech
(Beijing) Co., Ltd, China; anti-GAPDH (no: AF7021, Affinity); and
BCA Protein Assay Kit (no: P0012S, Beyotime Biotechnology, China).
materials and reagents were used in the study: Emodin (no.: A0044,
Chengdu Must Biotechnology Co., Ltd., China, purity≥98%); anti-MMP9
(no.: Ab38898, Abcam, U.K.); anti-NF-κB p65 (no.: #8242, CST,
USA); anti-IKKβ (no.: #2678, CST, USA); 2,3,5-triphenyl tetrazolium
chloride (no.: T8877-25G, Sigma, USA); hematoxylin and eosin (HE)
staining kit (no.:BP-DL017, SenBeiJia Biological Technology Co., Ltd.
China); HRP-labeled goat anti-rabbit IgG (H + L) (no.: A0208, Beyotime
Biotechnology, China); anti-MMP2 (no.: Ab181286), anti-occludin (no.:
Ab216327), anti-extracellular signal-regulated kinase [ERK1/2 (no.:
Ab184699)], and p-anti-ERK1/2 (no.: Ab214036)—Abcam, U.K.;
IL-6 antibody (no.: DF6087), HIF1A antibody (no./AF1009), claudin-5
(no./AF5216), and VEGFA antibody (no.: DF7470)—Affinity, China;
Alexa Fluor 555-labeled donkey anti-mouse IgG(H + L) (A0460), Triton
X-100 (no.: ST795), and antifade mounting medium (no.: P0126)—Beyotime
Biotechnology, China; neutral balsam (no.: G8590, Beijing Solarbio
Science & Technology Co., Ltd. China); RNA Easy Fast Tissue/Cell
Kit (no.: DP451), FastKing gDNA Dispelling RT SuperMix (no.: KR118),
and Talent qPCR PreMix (SYBR Green) (no.: FP209)—Tiangen Biotech
(Beijing) Co., Ltd, China; anti-GAPDH (no: AF7021, Affinity); and
BCA Protein Assay Kit (no: P0012S, Beyotime Biotechnology, China).
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