Co‐immunoprecipitation (Co‐IP) assays were performed as described previously (Zhang et al., 2019 (link)). About 0.5 g of agroinfiltrated leaf tissue was frozen in liquid nitrogen, ground to a fine powder, and then thawed in a plant protein extraction buffer (100 mM Tris–HCl, pH 8.8, 60% SDS, and 2% B‐mercaptoethanol) with protease inhibitor cocktail tablets (one tablet per 50 mL buffer). The mixture was centrifuged at 18 000 g for 10 min at 4 °C. Each supernatant (500 μL) was mixed with 45 μL of anti‐GFP‐conjugated agarose beads (Sigma) and incubated at 4 °C for 1.5 h with gentle shaking. Agarose beads were pelleted and washed three times with the Co‐IP buffer. The resulting pellets were boiled in SDS loading buffer. For the immunoblot, proteins were separated on 10% SDS‐PAGE gels (SurePAGE™, Genscript, M00652) through electrophoresis, and then transferred to NC membranes using eBlot™ L1 (Genscript, L00686C). The blots were probed with an anti‐HA (1 : 5000) and an anti‐GFP (1 : 5000), followed by an HRP‐conjugated secondary antibody (HUABIO, HA1006).
Ha1006
Manufactured by Huabio
Sourced in China
The HA1006 is a laboratory equipment designed for general-purpose use. It features a compact and durable construction. The core function of the HA1006 is to provide a controlled environment for various laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ha1001, by Huabio (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Surepage, by GenScript (1 mentions)
Eblot l1, by GenScript (1 mentions)
Ab124956, by Abcam (1 mentions)
Ab179461, by Abcam (1 mentions)
Tom20, by Cell Signaling Technology (1 mentions)
M1305 2, by Huabio (1 mentions)
Et1612 47, by Huabio (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Il 10, by Wuhan Servicebio Technology (1 mentions)
Bcl 2, by Huabio (1 mentions)
Bcl2 associated x (bax), by Huabio (1 mentions)
Ripa lysis buffer, by MultiSciences Biotech (1 mentions)
Cleaved caspase3, by Huabio (1 mentions)
11 protocols using ha1006
1
Co-Immunoprecipitation Assay of Plant Proteins
2
Immunoblotting Analysis of Protein Expression
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Cells were lysed by the RIPA lysis buffer (Beyotime, China) on ice. Protein concentrations were quantified by BCA protein assay (Beyotime, China). The immunoblots were probed with appropriate primary overnight at 4°C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h.
The blots were infiltrated with ECL (Bio-Rad, USA) and detected by ChemiDoc™MP Imaging System (Bio-Rad, USA). The densitometry of target bands was normalized to internal control β-actin. The antibodies are listed as follow up: Runx2 (1:1000, ET1612-47, Huabio, Hangzhou, China); OPN (1:1000, 0806–6, Huabio, Hangzhou, China); cleaved-caspase 3 (1:1000, 9664S, Cell Signaling Technology, USA); cleaved-caspase 9 (1:1000, 9509S, Cell Signaling Technology, USA); TOM20 (1:1000, 42406S, Cell Signaling Technology, USA); cytochrome c (1:1000, 12,963, Cell Signaling Technology, USA); T-JNK (1:1000, ab179461, Abcam, UK); p-JNK (1:1000, ab124956, Abcam, UK); T-c-JUN (1:1000; ET1608-3, Huabio, Hangzhou, China); p-c-JUN (1:1000; ET-1608-4, Huabio, Hangzhou, China); β-actin (1:3000, R1102-1, Huabio, Hangzhou, China); tubulin (1:3000, M1305-2, Huabio, Hangzhou, China); anti-rabbit, anti-mouse secondary antibody (1:3000, #HA1011, #HA1006, Huabio, Hangzhou, China).
The blots were infiltrated with ECL (Bio-Rad, USA) and detected by ChemiDoc™MP Imaging System (Bio-Rad, USA). The densitometry of target bands was normalized to internal control β-actin. The antibodies are listed as follow up: Runx2 (1:1000, ET1612-47, Huabio, Hangzhou, China); OPN (1:1000, 0806–6, Huabio, Hangzhou, China); cleaved-caspase 3 (1:1000, 9664S, Cell Signaling Technology, USA); cleaved-caspase 9 (1:1000, 9509S, Cell Signaling Technology, USA); TOM20 (1:1000, 42406S, Cell Signaling Technology, USA); cytochrome c (1:1000, 12,963, Cell Signaling Technology, USA); T-JNK (1:1000, ab179461, Abcam, UK); p-JNK (1:1000, ab124956, Abcam, UK); T-c-JUN (1:1000; ET1608-3, Huabio, Hangzhou, China); p-c-JUN (1:1000; ET-1608-4, Huabio, Hangzhou, China); β-actin (1:3000, R1102-1, Huabio, Hangzhou, China); tubulin (1:3000, M1305-2, Huabio, Hangzhou, China); anti-rabbit, anti-mouse secondary antibody (1:3000, #HA1011, #HA1006, Huabio, Hangzhou, China).
3
Western Blot Analysis of Myocardial Proteins
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Myocardial tissue and cell suspension was lysed by RIPA lysis buffer (MB-030-0050, Multi Sciences Biotech, Hangzhou, China). BCA Protein Assay Kit (23225, Thermo Fisher, USA) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (4-20%, ACE Biotechnology, Xiangtan, China), and the bands were transferred onto PVDF membranes (03010040001, Merck, USA). After blocked with 5% skimmed milk and washed with tris-buffered saline containing 0.1% tween-20 (TBST), protein bands were blotted with primary antibodies at 4° C overnight. After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (32209, Thermo Fisher). Quantifications of western blotting were measured with Fusioncapt advance software. Antibody information: Bax (HUABIO, USA, ET1603-34, 1:1000), Bcl2 (HUABIO, ET1702-53, 1:1000), Cleaved-caspase3 (HUABIO, ET1608-64, 1:1000), IL-10 (Servicebio, Wuhan, China, GB11108, 1:1000), TLR4 (Servicebio, GB11519, 1:1000), α-SMA (Servicebio, GB111364, 1:1000), MMP-9 (Abcam, Cambridge, UK, ab76003, 1:1000), Collagen I (Abcam, ab34710, 1:1000), GAPDH (CST, USA, 5174S, 1:1000). HRP-conjugated secondary antibody (HUABIO, HA1001, and HUABIO, HA1006, 1:50000).
4
Characterizing Intestinal Stem Cells and Proliferation
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For analysing the expression of Lgr5 (a marker of ISCs) and PCNA (a marker of proliferation cells) in the duodenum and crypt organoids, Immunohistochemistry (IHC), Immunofluorescence (IF) and Western Blot (WB) were performed. Briefly, for IHC/IF, the intestinal section samples were incubated with rabbit anti‐Lgr5 (1:50; HuaBio, customisation, Hangzhou, China) or mouse anti‐PCNA (1:200; Abcam, ab29, Cambridge, UK) overnight at 4°C, then incubated with fluorescein isothiocyanate conjugated goat anti‐rabbit secondary antibody (1:500; HuaBio, HA1001) or horseradish peroxidase (HRP) conjugated goat anti‐mouse secondary antibody (1:500; HuaBio, HA1006) for 1 h at 37°C. For WB, total proteins were extracted from the duodenal crypt and crypt organoids. After SDS‐PAGE separating, transferring to polyvinylidene fluoride membrane and skimmed milk blocking. The samples were incubated with rabbit anti‐Lgr5 (1:250) or rabbit anti‐β‐actin (1:50 000; Abclonal, AC026, Wuhan, China) overnight at 4°C, followed by incubation with the HRP‐conjugated goat anti‐rabbit (1:2000; Abclonal, AS014). For the semiquantitative assay, images were quantified and analysed using the Gel‐Pro software, and the grey analysis of target protein bands was normalised with β‐actin.
5
Histopathological Assessment of Liver Steatosis
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Mice were euthanized with carbon dioxide asphyxiation, and then liver tissues were removed immediately and washed with normal cold saline. The same portion of the liver samples in all mice was used for the histopathological examination. The rest fresh tissue samples were stored at −80°C for protein analysis. Livers were fixed overnight in a buffer solution containing 4% paraformaldehyde and then embedded in paraffin. The paraffin-embedded tissues were cut into 4 μm thickness sections and then stained with hematoxylin and eosin (H&E) or Oil Red O to assess hepatic steatosis. Additionally, to detect the protein expression of TNF-α and Nrf2 in liver tissues, immunohistochemistry (IHC) was applied. The primary antibodies anti-TNF-α (1: 100, #ab6671, Abcam, Cambridge, UK) and anti-Nrf2 (1: 200, #ab89443, Abcam), and IgG HRP-conjugated secondary antibodies, including goat anti-mouse (1: 100, HA1006) and goat anti-rabbit antibodies (1: 1000, HA1001, HuaBio, HangZhou, China) were applied for immunohistochemical staining. The preparations were photographed under an inverted microscope (Leica, Germany). The mean densitometry was quantitatively measured under a 400× magnification microscope for 6 microscopic fields randomly chosen by using Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and results were displayed as average.
6
Intestinal Crypt Protein Analysis
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Equal amounts of total protein extracted from the intestinal crypt cells, were electrophoresed on 12% polyacrylamide/sodium dodecyl sulfate gel and transferred onto polyvinylidene fluoride membranes. After being blocked for 45 min, the membranes were incubated with primary antibodies for 1 h. Subsequently, membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h after 3 times washing with PBS. Finally, the immunoreactivity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA, WBKLS0500). The antibodies used were as follows: anti-HSP60 (Abcam, ab46798, 1:1000), anti-LONP1 (Cell Signal Technology, 28020, 1:1000), anti-CDKN1A/p21 (Abclonal, A2691, 1:100), anti-Phospho-eIF2α-S51 (Abclonal, AP0692, 1:1000), anti-ClpP (Abcam, ab124822, 1:1000), anti-ATF4 (Abcam, ab216839, 1:1000), anti-ATF5(Abcam, ab184923, 1:1000), anti-CHOP(Cell Signal Technology, 2895 S, 1:1000), anti-TOM20 (Proteintech, 11802-1-AP, 1:1000), anti-WNT4 (Bio-Techne, MAB4751, 1:1000), anti-SIRT7 (Abclonal, A22735, 1:1000), anti-Actin (HUABIO, ET1702-67, 1:3000), HRP Conjugated Goat anti-Mouse IgG (HUABIO, HA1006, 1:3000) and HRP Conjugated Goat anti-Rabbit IgG (HUABIO, HA1001,1:3000).
7
Western Blot Analysis of APPL1 Protein
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For sample preparation, 106 cells were collected and lysed by 120 μL protein loading buffer (2x) and predenatured at 100°C for 10 min. The 12% SDS-PAGE was performed with 4 μL of cell protein samples, and the rest of the steps were performed according to the protocol of Western blot [21 (link), 22 (link)]. Antibodies used in the experiment included rabbit monoclonal antibody APPL1 (ab180140, Abcam), mouse monoclonal antibody α-tubulin (YM3035, Immunoway), and goat anti-rabbit/mouse HRP (HA1001, HA1006, HUABIO).
8
Peri-ischemic Area Protein Analysis
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Brain tissues in the peri-ischemic area and primary astrocyte were used for western blot analysis. The protein concentration was measured using a BCA protein kit (Epizyme). The proteins (30 μg/group) were separated into a 5-10% gradient SDS-PAGE gel and then transferred onto PVDF membranes. After blocking with 5% milk, the bands were incubated overnight at 4°Cwith primary antibodies, including anti-CD63 (1:1000, #sc-15363, Santa Cruz Biotechnology), anti-TSG101 (1:1000, #ab83, Abcam), anti-TNF (1:500, #sc-52746, Santa Cruz Biotechnology), anti-MMP3 (1:1000, # 17873-1-AP, ProteinTech Group), anti-NFκB p65 (1:1000, #8242T, Cell Signaling Technology), or anti-β-actin (1:1000, #66009, ProteinTech Group). Then the membranes were incubated with HRP-conjugated secondary antibody (1:5000, #HA1006 or #HA1001, HUABIO) at room temperature for 1 h. Visualization analysis was performed using an enhanced chemiluminescence substrate (#SQ201, Epizyme) and an imaging system (Bio-Rad, Hercules, CA).
9
Mycelia Protein Extraction and Western Blot Analysis
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The mycelia of strains were cultured in liquid PDA under 180 rpm at 28°C for 36 h, washed with ddH2O, and moved to MM-N medium under 80 rpm at 28°C for 2 h and 5 h. The mycelia were further collected and ground into powder in liquid nitrogen and then resuspended in 1 mL RIPA lysis buffer (EpiZyme, PC101) with 10 μL of a proteinase inhibitor cocktail (EpiZyme, GRF101). The lysates were incubated in ice for 30 min, resuspended with a Vortex-Genie every 10 min, and further centrifuged at 12,000 × g for 20 min at 4°C. The supernatant proteins were harvested. The proteins were analyzed by 10% SDS-PAGE followed by Western blotting with the primary antibodies being anti-GFP (rabbit, 1:5,000, Abways, AB0045), anti-α-H3K18ac (rabbit, 1:3,000, Beyotime, AF5617), anti-α-H3 (rabbit, 1:2,000, Cell Signaling Technology, 4499), and anti-β-tubulin (mouse, 10,000, Engibody, AT0003) and the secondary antibodies being HRP-labeled goat anti-rabbit lgG (H+L) (1:10,000, Abways, AB0101) and HRP-labeled goat anti-mouse lgG (H+L) (1:20,000, HUABIO, HA1006), respectively. Finally, the proteins were detected by an Omni-ECL Femto Light Chemiluminescence Kit (EpiZyme, SQ201) and analyzed by ImageJ.
10
Western Blot Protocol for Protein Analysis
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Cells were collected, washed twice with pre-cooled PBS, and lysed in lysis buffer (Cat No. P0013, Beyotime, Shanghai, China) supplemented with complete protease inhibitor cocktail tablets (Roche, Basel, Switzerland) at 4 °C for 20 min, and then mixed with 5 × loading buffer (Cat No. FD002, Fude, Zhejiang, China) and boiled for 30 min. Cell lysates were centrifuged at 13,000 rpm at 4 °C for 5 min. Supernatant were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins on gel were transferred to PVDF membranes (IPVH00010, Millipore, Ireland) using a wet apparatus (Bio-Rad, California, USA).80 (link) The membranes were incubated overnight with primary antibody dilutions. Next day, membranes were washed with TBST for three times and then incubated in secondary antibody dilutions (1:5000) for 1 h (Cat No. HA1006 or HA1001, HuaBio, Hangzhou, China). Exposure was performed with SignalBright Pro Chemiluminescent Substrate (Cat No. PK10011, Proteintech, Illinois, USA) following the manufacturer’s protocol.
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