Cells were lysed using RIPA buffer containing 1% sodium orthovanadate and protease inhibitors, transferred to 2 mL Eppendorf tubes, incubated for 30 minutes at 4 °C while rotating, and centrifuged. Protein concentrations were quantified using the Pierce BCA protein assay kit (23225, Thermo Fisher, Waltham, MA). Extracted proteins were loaded into 30 μL wells of 4–15% Immobilion Protean gels (446–1083, Bio-Rad, Hercules, CA), electrophoresed then transferred to Immobilion PVDF membranes. Membranes were blocked for one hour in Licor blocking buffer (927–40,000, LI-COR, Lincoln, NE), incubated overnight at 4 °C with the appropriate primary antibodies, washed four times with 0.1% Tween20 in PBS (PBST) and finally incubated for one hour with the appropriate secondary antibodies (Licor IRDye 680/800). Experiments were performed in triplicate for each condition for the following proteins: H3K9ac (Millipore, #06–942), H3K9me3 (Abcam, Cambridge, MA, ab8898), H3K27me3 (Millipore, #07–449), lamin A/C (Cell Signaling Technology, Danvers, MA, #2032), lamin B1 (Abcam, ab16048), and lamin B2 (Abcam, ab8983). β-actin (Sigma Aldrich, A2103) was used as the loading control. The blots were imaged and quantified using the Odyssey platform and software (LI-COR).
Lamin b2
Manufactured by Abcam
Sourced in United Kingdom
Lamin B2 is a nuclear envelope protein that is a component of the nuclear lamina. It plays a structural role in maintaining the integrity and organization of the nuclear envelope.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Blocking buffer, by LI COR (1 mentions)
H3k9ac, by Merck Group (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
H3k9me3, by Abcam (1 mentions)
Lamin b1, by Abcam (1 mentions)
H3k27me3, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Nup153, by Abcam (1 mentions)
Lap2β, by BD (1 mentions)
Reagents for cell culture, by Thermo Fisher Scientific (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified, by Merck Group (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Tubulin, by Abcam (1 mentions)
Secondary antibody conjugated to hrp, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
6 protocols using lamin b2
1
Western Blot Analysis of Histone Modifications and Lamins
2
Immunocytochemistry and Western Blot Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used for immunocytochemistry and Western blot analysis: rabbit polyclonal antibodies against lamin B1, Tpr, importin α7 (Abcam, Cambridge, MA), pHIST3 (serine 10), MAP-2 (Merck Millipore, Darmstadt, Germany), actin, βIII-tubulin (Sigma-Aldrich, Milano, Italy), pERK, pCREB (Cell Signaling, Beverly, MA), lamin A/C (Santa Cruz Biotechnology, Dallas, TX), synaptophysin (SySy, Gottingen, Germany), and drebrin (Enzo Life Sciences, Farmingdale, NY); and mouse monoclonal antibodies against Nup153, lamin B2, CREB (Abcam), nuclear pore complex (mAb414; Covance, Princeton, NJ), Lap2β (BD Biosciences, East Rutherford, NJ), Tau (Tau-1; Merck Millipore), ERK (Cell Signaling), importin β (Sigma-Aldrich), and Alexa 488–conjugated Tuj1 (Covance). For Western blot analysis, horseradish peroxidase (HRP)–conjugated secondary antibodies (Bio-Rad, Hercules, CA) were used for detection. For immunofluorescence analyses, Alexa fluorophore–conjugated anti-mouse, anti-rabbit, and anti-rat antibodies from Invitrogen (Thermo Fisher Scientific, Waltham, MA) were used. Unless otherwise specified, general reagents and chemicals were from Sigma-Aldrich, and reagents for cell cultures were from Invitrogen.
3
Modulation of β-catenin Signaling in Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C184 stem cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma). CGR8-ES cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 20 mM L-glutamine, 50 mM β -mercaptoethanol, and 100-units/ml leukemia inhibitory factor (ESGRO-Chemicon). Chir99021 (5 μM) together with CDNG1 (10 μM), CDMG1 (10 μM), or CDMG2 (10 μM) were added into media. After 3-h treatment, cells were harvested and incubated in ice-cold radio immunoprecipitation assay (RIPA) cell lysis buffer containing protease inhibitors. The proteins were separated by SDS-polyacrylamide gel electrophoresis, probed with antibodies against β -catenin (Santa Cruz Biotechnology, 1:200), β -actin (Sigma, 1:500), Tubulin (Abcam, 1:500), and lamin B2 (Abcam, 1:500) and detected by horseradish peroxidase–conjugated antibodies. Image J measured the band intensity for quantification. C184 stem cells were treated with Chir99021 (5 μM) together with CDNG1 (10 μM), CDMG1 (10 μM), or CDMG2 (10 μM). After 3-h treatment, cells were subjected to immunostaining of β -catenin antibodies and 4 ,6-diamidino-2-phenylindole (DAPI) staining. Secondary antibodies used include AlexaFluor Alexa 488 (Invitrogen).
4
Protein Analysis via Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole protein was extracted with cell lysis buffer. After that, 30 μg protein per lane was fractionated on 10% SDS-PAGE, and then transferred onto PVDF membrane. After that, the membranes were incubated with nonfat milk. Primary antibodies against FN, Col IV, ALK7, Nrf2, lamin B2, HO-1, NQO-1, and β-actin (Abcam) and secondary antibody conjugated to HRP (1: 3000; Abcam) were, respectively, added to the membranes. Finally, chemiluminescent substrate was then used to visualize the bands.
5
Protein and RNA Profiling of Tissue Samples
6
Nuclear and Cytoplasmic Protein Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nuclear and cytoplasmic protein fractions from HaCaT cells were extracted using NE-PER Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA), according to the protocol from the manufacturer. The protein concentration was evaluated using Bradford assays. Subsequently, 20 µg of the total proteins were resolved using 10% SDS-PAGE electrophoresis and transferred to PVDF membranes (Merck Millipore, Carrigtwohill, Ireland). The membranes were blocked in a 5% skim milk solution, then incubated with the primary antibodies, followed by secondary antibody horseradish peroxidase (HRP)-conjugated anti-IgG. The primary antibodies included anti-NF-κB p65 (#8242S, Cell Signaling Technology, Danvers, MA, USA), p-IKK (#2694S, Cell Signaling Technology), IκB-α (#4814S, Cell Signaling Technology), lamin B2 (ab151735, Abcam, Cambridge, UK), and β-actin (A1978, Sigma-Aldrich, St. Louis, MO, USA). The HRP-conjugated goat anti-rabbit and goat anti-mouse IgG secondary antibodies were procured from Sigma-Aldrich (St. Louis, MO, USA). All blots were visualized using ECL reagents and ChemiDoc imaging systems (BioRad, Hercules, CA, USA). The relative intensities of the protein bands were determined using Gel-Pro analyzer software (version 3.1, Media Cybernetics, Rockville, MD, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!