Mouse brain tissues were lysed with ice-cold RIPA buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA; 8340) and 2% phosphatase inhibitors (Abcam, Cambridge, MA, USA; ab201112). The protein concentrations were measured by Bradford assay (Beyotime Institute of Biotechnology; P0010). Equal amounts of protein were separated on 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then incubated in 5% skim milk for 1 h and then incubated overnight at 4°C with the primary antibodies (Supplementary Table 2 ). Membranes were washed with TBS-T 3 times for 10 min each and then were incubated with horseradish peroxidase–labeled secondary antibodies (HRP 1:10000, Thermo Fisher Scientific, Waltham, MA, USA) for 1.5 h at room temperature. After washing 3 times, bands were detected using ECL (EMD Millipore). Protein band intensity was quantified using ImageJ software.
Phosphatase inhibitor
Manufactured by Abcam
Sourced in United States, China
Phosphatase inhibitors are chemical compounds used in biological research to prevent the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from proteins, affecting their function and signaling pathways. These inhibitors are employed to maintain the phosphorylation state of proteins, allowing for the study of phosphorylation-dependent processes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Protease inhibitor, by Abcam (2 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Bradford assay, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Sds polyacrylamide gel, by Beyotime (1 mentions)
Rabbit anti lc3b, by Abcam (1 mentions)
Chicken anti albumin, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Aldh1, by Cell Signaling Technology (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Rotor gene sybr green pcr kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rotor gene q real time pcr cycler, by Qiagen (1 mentions)
10 protocols using phosphatase inhibitor
1
Western Blot Analysis of Mouse Brain Proteins
2
Zebrafish Intestinal Protein Analysis
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Zebrafish intestines or larval zebrafish were lysed with ice-cold RIPA lysis buffer mixed with 1 mM PMSF and phosphatase inhibitors (Abcam, USA). Equivalent amounts of total protein were loaded into a 12% SDS-PAGE for electrophoresis and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking nonspecific binding with 5% non-fat dry milk in PBS, the PVDF membrane was incubated with primary antibodies, i.e., antibodies against β-actin (CMCTAG, AT0544, 1:1000), HIF-1α (Bioworld, BS3514, 1:1000), TLR4 (Cell Signaling Technology, 14358S, 1:1000). The blots were developed using horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Health, 1:3000) and the ECL-plus system.
3
Protein Expression Analysis in Rat Brain
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Rats were administered 4% chloride hydrate and sacrificed. Brain tissues were removed and
separated on ice. Peri-injury cortical tissues, which contained the target protein, were
immediately frozen at –80°C before use. The sheared tissues were then dissolved by RIPA
lysis buffer (Beyotime, Shanghai, China) and phosphatase inhibitors (Abcam, Cambridge, UK)
and centrifuged at 13,000 rpm at 4°C for 30 min. The protein concentration of the
collected supernatant was tested by a PierceTM BCA Protein Assay Kit
(Thermo-Fisher Scientific, Loughborough, UK). The resulting samples were separated by
electrophoresis on 10% SDS-polyacrylamide gels (Beyotime, Shanghai, China). After transfer
to PVDF membranes (Millipore, Bedford, MA, USA), 5% nonfat milk was used to block the
membranes at room temperature for 2 h. The membranes were then incubated with the
following primary antibodies overnight at 4°C: rabbit anti-BNIP3 L (1:400, Abcam), rabbit
anti-LC3B (1:1000, Abcam), rabbit anti-SQSTM1 (1:1000, Abcam), chicken anti-Albumin
(1:1000, Abcam), and rabbit anti-GAPDH (1:10000, Abcam). A corresponding horseradish
peroxidase (HRP)-conjugated secondary antibody was used to react with the appropriate
primary antibody. Protein bands were developed in an enhanced chemiluminescence system
(ECL, Pierce, Waltham, MA, USA) and then analyzed by using ImageJ software.
separated on ice. Peri-injury cortical tissues, which contained the target protein, were
immediately frozen at –80°C before use. The sheared tissues were then dissolved by RIPA
lysis buffer (Beyotime, Shanghai, China) and phosphatase inhibitors (Abcam, Cambridge, UK)
and centrifuged at 13,000 rpm at 4°C for 30 min. The protein concentration of the
collected supernatant was tested by a PierceTM BCA Protein Assay Kit
(Thermo-Fisher Scientific, Loughborough, UK). The resulting samples were separated by
electrophoresis on 10% SDS-polyacrylamide gels (Beyotime, Shanghai, China). After transfer
to PVDF membranes (Millipore, Bedford, MA, USA), 5% nonfat milk was used to block the
membranes at room temperature for 2 h. The membranes were then incubated with the
following primary antibodies overnight at 4°C: rabbit anti-BNIP3 L (1:400, Abcam), rabbit
anti-LC3B (1:1000, Abcam), rabbit anti-SQSTM1 (1:1000, Abcam), chicken anti-Albumin
(1:1000, Abcam), and rabbit anti-GAPDH (1:10000, Abcam). A corresponding horseradish
peroxidase (HRP)-conjugated secondary antibody was used to react with the appropriate
primary antibody. Protein bands were developed in an enhanced chemiluminescence system
(ECL, Pierce, Waltham, MA, USA) and then analyzed by using ImageJ software.
4
Western Blot Analysis of Stem Cell Markers
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Cells and tissues were harvested, and protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) with phosphatase inhibitors (Abcam, Cambridge, UK). Then, the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The samples were blocked with 2% BSA and incubated overnight at 4°C with primary antibodies ALDH1 (#54135, 1:1000; Cell Signaling Technology [CST], Danvers, MA, USA), CD44 (#37259, 1:1000; CST, Danvers, MA, USA), NANOG (#4903, 1:1000; CST), Oct4 (#2750, 1:1000;CST), β-actin (#4970, 1:1000; CST), KDM7A (Thermo Fisher Scientific, New York, CA, USA). The antirabbit IgG-HRP (#7074, 1:2000; CST) was applied as the secondary antibody. The bands were determined using a Bio-Rad GelDoc XR (Bio-Rad, Hercules, CA, USA).
5
Tissue Harvesting for Protein Analysis
6
Spinal Cord Injury Protein and RNA Analysis
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0.5 cm long segments of spinal cord tissue centered on the lesion site were collected at various time points after injury following PBS perfusion of the animals (different animals from BMS group) and flash‐frozen on dry ice. For proteins, the tissues were lysed in RIPA buffer (10 mmol/L sodium phosphate pH 7.2, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% sodium deoxycholate, 1% NP‐40, 0.1% sodium dodecyl sulfate) supplemented with protease inhibitors (Pierce tablets) and phosphatase inhibitors (BioVision). Proteins were resolved on SDS‐PAGE gels before being transferred onto nitrocellulose membranes using the Trans‐Blot® Turbo™ system (Bio‐Rad). Antibodies for the Western blot detection included rabbit anti‐CNPase (1:5000; Cell signaling), mouse anti‐NF200 (1:5000; Sigma), rabbit anti‐PI3Kγ (1:500; Santa Cruz), and mouse anti‐β‐tubulin (1:60 000; Sigma).
RNAs were extracted using TRIzol (Thermo Fisher) following the manufacturer's instructions. RT‐PCR was performed with the Omniscript RT kit (Qiagen) followed by qPCRs performed using the Rotor‐Gene SYBR® Green PCR Kit, and the Rotor‐Gene Q real‐time PCR cycler (Qiagen). Each gene expression levels were determined using a standard curve for that gene, and the data were normalized to β‐actin expression levels. The primers used are listed in the TableS2 .
RNAs were extracted using TRIzol (Thermo Fisher) following the manufacturer's instructions. RT‐PCR was performed with the Omniscript RT kit (Qiagen) followed by qPCRs performed using the Rotor‐Gene SYBR® Green PCR Kit, and the Rotor‐Gene Q real‐time PCR cycler (Qiagen). Each gene expression levels were determined using a standard curve for that gene, and the data were normalized to β‐actin expression levels. The primers used are listed in the Table
7
Extraction and Quantification of Proteins from EAE Mouse Tissues
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EAE mice were deeply anesthetized at 30 dpi with Ketamine (215 mg/kg) and Xylazine (43 mg/kg) and subsequently perfused with phosphate buffered saline (PBS). Sensorimotor cortex and whole lumbar spinal cord (SC) were extracted and immediately stored on dry ice. Tissue was then homogenized in RIPA buffer (10 mM sodium phosphate buffer pH 7.2, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Santa Cruz, Dallas, TX) and phosphatase inhibitors (Biovision, Milpitas, CA) and incubated at 4 °C for 30 min on a rocker. The samples were then centrifuged at 4 °C for 15 min at 13,200RPM. The resulting supernatant was transferred to a separate tube from which protein concentrations were determined using the DC™ protein assay (Biorad, Hercules, CA).
8
Protein Extraction from Tissue Samples
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Tissues were weighed and then homogenized in lysis buffer (20 mM Tris (pH 7.8), 17 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% (w/v) glycerol, 1 mM EDTA, supplemented with protease inhibitor (Roche, Mannheim, Germany) and phosphatase inhibitor (Abcam, Cambridge, UK)). The ratio of tissue weight (mg) to lysis buffer volume (µL) was maintained at 1:10. Homogenization was performed by incubating the samples for 4 h at 4 °C with continuous agitation. After, samples were centrifuged at 13,000 rpm for 15 min, and the supernatants were collected. Total protein quantification was performed using the Bicinchoninic acid assay (Pierce, Waltham, MA, USA). All samples were run in duplicates, and absorbance was measured at 575 nm with an iMarkTM Microplate Absorbance Reader.
9
Western Blot Analysis of WJ-MSCs
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Following different experimental treatments, WJ-MSCs were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (Abcam, Cambridge, UK). Protein concentration was quantified using the Bradford assay. Western blotting was performed as per standard protocols. The primary and secondary antibodies used were as follows: anti-VTN, anti-p53, anti- p65, anti-GAPDH (all from Santa Cruz Biotechnology, Inc. Dallas, TX, USA), and horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling Technology, Inc., Danvers, MA, USA). Immunoreactivity was detected using chemiluminescent femtoLucent PLUS-HRP (G-Biosciences, Maryland Heights, MO, USA). Chemiluminescent blots were imaged using G:Box (Syngene, Frederick, MD, USA).
10
Autophagy regulation by KLF5 and Bcl2
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3-methyladenine (3-MA, 189490) and Bafilomycin A1 (BAFA1, 196000) were purchased from EMD Millipore (Darmstadt, Germany). Docetaxel and PF4708671 were from Selleck Chemicals (Houston, TX, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RIPA buffer was acquired from Cell Signaling Technology (Danvers, MA, USA), protease inhibitor and phosphatase inhibitor were from Abcam (Cambridge, MA, USA). Roche (Mannheim, Germany). Primary antibodies against LC-3 I/II, Beclin1, ATG3, ATG5, ATG7, mTOR, p-mTOR (Ser2448), ACC, p-ACC (Ser79), p-AMPK(Thr172), and AMPK were acquired from Cell Signaling Technology and HDAC3 was from Abcam (Cambridge, MA, USA). PVDF membrane was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The siKLF5 #1, siKLF5 #2 siRNAs, siBeclin1 #1, siBeclin1 #2 siRNAs, siBcl2 #1, siBcl2 #2 siRNAs and the scrambled RNA, which used as shcontrol were obtained from RiboBio (Guangzhou, China). PLKO.1 lentiviral vectors encoding short hairpin RNA (shRNA) targeting non-specific control (NC) or human KLF5 (sh710: 5′-GGTTACCTTACAGTATCAACA-3′) were constructed by GenPharma (Shanghai, China).
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