Protein isolation and Western blotting analysis of cell lysates were performed as previously described [12 (link),13 (link)]. For the analysis of nuclear NFκB p65, NE-PER nuclear and cytoplasmic extraction reagents (Pierce, USA) were used to separate cytoplasmic and nuclear fractions. Membranes were first probed with primary antibodies for phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, NFκB p65, phospho-p65 (Ser536), EMMPRIN, and GAPDH (Cell Signaling Technology, USA), and lamin B (Sigma-Aldrich, USA), and then incubated with anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology, USA), followed by incubation with antibody labeled with far-red fluorescent Alexa Fluor 680 dye. All signals were detected by the Odyssey Infrared Imaging System (LI-COR, USA) and data were normalized by GAPDH or lamin B levels (for NFκB p65).
Lamin b
Manufactured by Merck Group
Sourced in United States
Lamin B is a lab equipment product manufactured by Merck Group. It is a structural protein found in the cell nucleus that plays a crucial role in the organization and maintenance of the nuclear envelope.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti rabbit or anti mouse secondary antibodies, by Cell Signaling Technology (1 mentions)
Emmprin, by Cell Signaling Technology (1 mentions)
Lamin b, by LI COR (1 mentions)
Gapdh, by LI COR (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
Creg1, by Abcam (1 mentions)
Antibodies against β actin, by Abcam (1 mentions)
Biotinylated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Nupagetm 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hmgb1 antibody, by Abcam (1 mentions)
Nf κb p65 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic protein extraction reagents, by Thermo Fisher Scientific (1 mentions)
Microplate bca protein assay kit reducing agent compatible, by Thermo Fisher Scientific (1 mentions)
9 protocols using lamin b
1
Western Blotting Analysis of Cell Signaling
2
Western Blot Analysis of Cellular Proteins
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Tissues were homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Thermo). Cells were lysed in either RIPA buffer or extraction buffer provided by a Nuclear Extraction Kit (Abcam) to obtain total proteins or nuclear proteins. Western blotting was performed as described previously [25 (link)]. Primary antibodies against Creg1 (Abcam), phosphorylated (p)- NF-κB (p65), NF-κB, p-IκBα and IκBα (Cell Signaling Technology, Danvers, MA) were used with 1:1000 dilution. Antibodies against β-actin (Abcam) and Lamin B (Sigma) were used as a loading control for total proteins or nuclear proteins, respectively. After blotting, films were scanned and the bands of interest were quantified by densitometry using Image J software (National Institutes of Health).
3
Protein Extraction and Western Blotting
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Cell extracts were lysed on ice with radio immunoprecipitation assay-buffer (Thermo Fisher Scientific, Rockford, IL, USA). Nuclear and cytoplasm protein fractions were isolated with NE-PER nuclear and cytoplasmic protein extraction reagents (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentrations were determined by microplate BCA protein assay kit-reducing agent compatible (Thermo Fisher Scientific, Rockford, IL, USA). For Western blotting, 30 μg of protein per lane were loaded on to NuPAGETM 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA).
Primary polyclonal antibody against mouse IL-33 was purchased from R&D Systems, (Minneapolis, MN, USA), Toll-like receptor 4 monoclonal antibody (mouse specific) was purchased from Cell Signaling (Danvers, MA, USA). HMGB1 antibody was purchased from Abcam (Cambridge, MA, USA), NF-κB p65 monoclonal antibody was purchased from Cell Signaling (Danvers, MA, USA). The biotinylated secondary antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
The bands were detected using Plus-ECL enhanced chemiluminescence kit (PerkinElmer, MA, USA). Membranes were stripped and reprobed for β-actin or LaminB (Sigma Aldrich, MO, USA) that served as a loading control.
Primary polyclonal antibody against mouse IL-33 was purchased from R&D Systems, (Minneapolis, MN, USA), Toll-like receptor 4 monoclonal antibody (mouse specific) was purchased from Cell Signaling (Danvers, MA, USA). HMGB1 antibody was purchased from Abcam (Cambridge, MA, USA), NF-κB p65 monoclonal antibody was purchased from Cell Signaling (Danvers, MA, USA). The biotinylated secondary antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
The bands were detected using Plus-ECL enhanced chemiluminescence kit (PerkinElmer, MA, USA). Membranes were stripped and reprobed for β-actin or LaminB (Sigma Aldrich, MO, USA) that served as a loading control.
4
Western Blot Analysis of Signaling Proteins
5
Western Blot Analysis of Cellular Signaling
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Cells were lysed with 100 mM phenylmethanesulfonyl fluoride, and the protein extracts were denatured and loaded onto a 10% SDS-PAGE gel. To measure IκB-α phosphorylation and nuclear p65 expression levels, NE-PER nuclear and cytoplasmic extraction reagents were used to isolated protein from the cytoplasm or nuclear. Then, western blot analysis were performed as we reported previously (Huang et al., 2012 (link); Cao et al., 2014 (link)). Briefly, membranes were first incubated with primary antibodies for Lamin B (Sigma-Aldrich, St. Louis, MO), EMMPRIN (Life Technologies, USA), MMP-9, p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-IκB-α, p65, or β-actin (Cell Signaling Technology, Boston, MA) then incubated with far-red fluorescent secondary antibodies (Life technologies, USA). All signals were conducted by the Odyssey imaging system (Li-cor, USA).
6
Mitochondrial Complexes Profiling in Macrophages
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Protein expression in stimulated macrophages was determined by western blot using 20 μg of total protein per sample3 (link). Separation into cytoplasmic and nuclear fractions was performed using 0.4 M NaCl and 0.1% Nonidet P-40 (NP-40) in extraction buffer57 (link). SDS immunoblots (IBs) were performed to evaluate steady-state protein levels of structural subunits of mitochondrial complexes assessed by native gels. Antibodies in this study are as follows: anti ACO1/2, anti IDH1, anti SDHB, anti SDHA, anti NDUFS1, anti MTCO1, anti ATP5A, anti NDUFS8, anti total OXPHOS (Complex V, ATP5A subunit; Complex IV, COXII subunit; Complex III, UQCRC2 subunit; Complex II, SDHB; Complex I, NDUFB9 subunit) and anti Phospho-S293 PDHE1-alpha were from Abcam; anti-FECH antibody was from Proteintech; anti phospho-, total PDK1 and anti HIF1α were from Novus Biologicals. For loading control anti CS, anti TOM20, Lamin B and anti β-actin were used (respectively from Sigma, Santa Cruz, Abcam, and Millipore).
7
Chondrocyte Inflammatory Response Inhibition
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Kirenol (purity > 98%) was obtained from Solarbio (Beijing, China), and Recombinant human IL-1β was purchased from PeproTech (NJ, USA). Collagenase type II and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (MO, USA). Primary antibodies against collagen II, COX-2 and iNOS were obtained from Abcam (Cambridge, UK). Primary antibodies against p65, IkBα, AKT and p-AKT were obtained from Cell Signaling Technology (MA, USA). β-actin and Lamin B antibodies were obtained from Sigma Aldrich (St Louis, MO, USA). Alexa Fluor®488 labeled and Goat Anti-Rabbit IgG second antibody were purchased from Jackson ImmunoResearch (West Grove, PA). The 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Shanghai, China). The cell culture reagents were obtained from Gibco (NY, USA).
8
Western Blot Antibody Panel for Neurodegenerative Markers
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We used commercially available antibodies for the following antigens: Aβ (1:500, clone 6E10, BioLegend), γH2Ax (1:1000, #2577), H2Ax (1:1000, #7631), Histone H3 (1:1000, #9717), Caspase3 (1:1000, #9662), cleaved Caspase-3 (1:1000, #9661), α-Tubulin (1:1000, #2144, Cell Signaling Technology, MA), NeuN (1:500, EPR12763, abcam, UK), MAP2 (1:1000, clone Ap20, BD Biosciences, NJ), AT8 (1:1000, MN1020), AT180 (1:1000, MN1040), AT100 (1:1000, MN1060), ZO-1 (1:500, 40-2200), Tau5 (1:1000, AHB0042, Thermo Fisher Scientific, MA), γH2Ax (1:1000, host mouse, clone JBW301,#05-636), Tau-1 (1:1000, clone PC1C6, MAB3420), Olig2 (1:500, AB9610), β-actin (1:10000, A5441), H3K9me3 (1:1000, 05-499), Tau oligomeric (T22, 1:1000,#ABN454, millipore, CA), LaminB (1:1000, M-20, sc-6217), β Tubulin (1:1000, sc-5274), GAPDH (1:5000, FL-335, sc-25778, santa cruz biotech.), GFAP (1:500, G 3893, Merck, DE), mouse tau (1:1000, 012-26963), and Iba1 (1:500, 013-27691, FUJIFILM Wako Chemical Coporation, JP); mouse, rabbit and goat IgG HRP-conjugated (Jackson ImmunoResearch, PA).
9
HUVEC Protein Lysate Analysis
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Equal amounts of protein lysates from HUVECs was harvested and lysed by SDS-PAGE and transferred to PVDF membrane (Merck Millipore, IPVH00010), then subjected to western blotting analysis: Membranes were blocked with 5% (v/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) and incubated with primary antibodies overnight at 4℃. The proteins were visualized using an Image Quant LAS 4000 (GE Healthcare) system, and the secondary antibodies are: goat anti-rabbit HRP (Bio-Rad, 1706515), goat anti-mouse HRP (Bio-Rad, 1706516).
For nuclear Nrf2 accumulation assays, HUVECs were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
The primary antibodies used to probe the membranes included Nrf2 (Santa Cruze Biotechnology, sc365949), Bax (Abcam, ab32503) and Bcl-2 (Abcam, ab59348) and GAPDH (CST, 5174), Lamin B (CST, 12,586), Keap1 (CST, 4678), c-Caspase-3 (CST, 9661), HDAC3 (CST, 85057), eNOS (CST, 32027), acetylated-lysine (CST, 9441), 3-NT (Millipore, 05–233), Nox4 (Origene, TA349083).
For nuclear Nrf2 accumulation assays, HUVECs were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China).
The primary antibodies used to probe the membranes included Nrf2 (Santa Cruze Biotechnology, sc365949), Bax (Abcam, ab32503) and Bcl-2 (Abcam, ab59348) and GAPDH (CST, 5174), Lamin B (CST, 12,586), Keap1 (CST, 4678), c-Caspase-3 (CST, 9661), HDAC3 (CST, 85057), eNOS (CST, 32027), acetylated-lysine (CST, 9441), 3-NT (Millipore, 05–233), Nox4 (Origene, TA349083).
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