Human recombinant FSP1, which is 93% identical to mouse FSP1, was generated as previously described7 (link). Rabbit polyclonal anti-human FSP1 antibody (1:5000 dilutions), mouse anti-human FSP1 monoclonal antibody (1:2000 dilutions), and rabbit polyclonal anti-mouse FSP1 (1:5000 dilutions) were generated as previously described5 (link)–7 (link). The goat polyclonal synaptopodin antibody (P-19) (sc-21537) (1:5000 dilutions) was purchased from Santa Cruz Biotechnology (US). Antibodies against AKT (4691S) (1:1000 dilutions), pAKT (4060S) (1:1000 dilutions), Lamin A/C (2032S) (1:1000 dilutions), and cleaved caspase-3 (9661 for western blot, 9664 for immunohistochemistry) (1:1000 dilutions) were purchased from Cell Signaling Technology (US). An antibody against Nrf2 (BS1074R) (1:1000 dilutions) was purchased from Bioss (US), and an antibody against β-actin (ab8227) (1:1000 dilutions) was purchased from Abcam, (UK). Nonspecific reactions were blocked with blocking reagent (X0909), purchased from Agilent (US). Cisplatin was purchased from SIGMA-Aldrich (US).
Lamin a c 2032
Manufactured by Cell Signaling Technology
Sourced in United States
Lamin A/C (2032) is a primary antibody product from Cell Signaling Technology. It recognizes Lamin A and Lamin C, integral proteins of the nuclear lamina.
Automatically generated - may contain errors
Lab products found in correlation
Pakt 4060, by Cell Signaling Technology (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Protein fractionation kit, by Thermo Fisher Scientific (1 mentions)
Anti aaas ta808612, by OriGene (1 mentions)
α tubulin 3873s, by Cell Signaling Technology (1 mentions)
Gapdh g8795, by Merck Group (1 mentions)
Actin a2066, by Merck Group (1 mentions)
Lamp1 ab25630, by Abcam (1 mentions)
Whatman, by Cytiva (1 mentions)
Ha sc 805, by Santa Cruz Biotechnology (1 mentions)
Flag f3165, by Merck Group (1 mentions)
P62 h00008878 m01, by Abnova (1 mentions)
Cyp2e1 ab28146, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Acetic acid, by Merck Group (1 mentions)
7 protocols using lamin a c 2032
1
Generating Antibodies for FSP1 Analysis
2
Quantitative Protein Analysis in Cellular Samples
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Protein was isolated from cells and tissue samples using radioimmunoprecipitation assay buffer enriched with protease and phosphatase inhibitors (Roche, Basel, Switzerland). Equal amounts of protein were loaded and separated on sodium dodecyl sulphate-acrylamide gels and then incubated with specific antibodies. The following primary antibodies were used: pAKT (4060S), AKT (2967S), pFOXO1 (9461S), FOXO1 (2880S), Lamin A/C (2032S) and PEPCK (12940S) from Cell Signaling Technology (Danvers, MA, USA). GAPDH (SC-25778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and G6P (ab83690) was from Abcam (Cambridge, UK). The protein levels were detected using Pierce ECL western blotting substrate (Waltham, MA, USA) following the standard western blot procedure, as described previously.10 (link) Images were captured with a UVP Bio-Spectrum 600 imaging system (Ultra-Violet Products Ltd. Cambridge, UK).
3
Protein Fractionation and Western Blot Analysis
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Protein aliquots were extracted from fibroblasts and autoptic tissues using denaturating (SDS) and reducing (Beta-mercaptoethanol) agents. Independent protein extractions were performed from cortical adrenal gland avoiding the adrenal medulla. Protein lysates of samples overexpressing wild-type and mutated Aladin were separated on a 4–12% polyacrylamide gel and blotted on a nitrocellulose membrane (Whatman). Subcellular fractions of tissues (autoptic cortical adrenal gland) were obtained using Protein Fractionation Kits (Thermofisher).
Samples were probed with the following antibodies: Anti-AAAS TA808612 (Origene) mouse (1:1000), Actin A2066 (Sigma) rabbit (1:1200), Lamp1 AB25630 (Abcam) mouse (1:800), Lamin a/c 2032S (Cell signaling) rabbit (1:800–1000), α-tubulin 3873S (Cell signaling) mouse (1:800–1:1000), GAPDH G8795(Sigma) rabbit (1:1000), PKA EP2606Y (Abcam) rabbit (1:5000) as loading and fractionating controls.
Samples were probed with the following antibodies: Anti-AAAS TA808612 (Origene) mouse (1:1000), Actin A2066 (Sigma) rabbit (1:1200), Lamp1 AB25630 (Abcam) mouse (1:800), Lamin a/c 2032S (Cell signaling) rabbit (1:800–1000), α-tubulin 3873S (Cell signaling) mouse (1:800–1:1000), GAPDH G8795(Sigma) rabbit (1:1000), PKA EP2606Y (Abcam) rabbit (1:5000) as loading and fractionating controls.
4
Comprehensive Antibody Usage in Autophagy Analysis
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The following antibodies were used: p62 (H00008878-M01) from Abnova (Taipei, Taiwan), Beclin-1 (sc11427), FXR (sc13063), and HA (sc805) from Santa Cruz Biotechnology (Santa Cruz, CA), and Flag (F3165) from Sigma (St. Louis, MO), serine 473 phosphorylated Akt (4058), Akt (9272), serine 9 phosphorylated GSK3β (5558S), GSK3β (5676S), serine 253 phosphorylated FoxO3a, FoxO3a (2497), GAPDH (2118), and Lamin A/C (2032) from Cell Signaling Technology (Beverly, MA), and CYP2E1 (ab28146) from Abcam (Cambridge, MA). The rabbit polyclonal anti-LC3B antibody was described previously [34] (link). The secondary antibodies used for immunoblotting analysis were HRP-conjugated goat anti-mouse (115-035-062), rabbit (111-035-045), and rat (111-035-143) and a Dylight 549 conjugated goat anti-rabbit (111-505-144) antibody from Jackson ImmunoResearch (West Grove, PA) or an HRP-conjugated goat anti-rabbit antibody (31460) from Thermo Fisher Scientific (Waltham, MA). Ethanol was from Pharmaco (Brookfield, CT). All other chemicals were from Sigma-Aldrich, (St. Louis, MO), Thermo, Fisher Scientific (Waltham, MA), Invitrogen (Carlsbad, CA), or EMD Millipore (Billerica, MA).
5
Investigating Anti-Inflammatory Effects of Dexamethasone and Carrageenan in RAW264.7 Cells
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RAW264.7 mouse monocyte/macrophage cells (No. TIB-71) were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA), and the DMEM medium and fetal bovine serum (FBS) were purchased from Gibco (a subsidy of Thermo Fisher Scientific, Waltham, MA, USA). Dimethylsulfoxide (DMSO), lipopolysaccharide (LPS, Escherichia coli 015B), Evans blue, acetic acid, dexamethasone (DEXA), and γ-carrageenan (Carr) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). NF-κB p65 (#8242), phospho-IκB-α (#2859), IκB-α (#4814), Lamin A/C (#2032), and β-actin (#4967) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). iNOS antibody (NB300-605) was obtained from Novus Biologicals (Littleton, CO, USA). COX-2 antibody (ab15191) was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit and goat-mouse secondary antibodies were obtained from EMD Millipore Corporation (Billerica, MA, USA).
6
Comprehensive Antibody Mapping for Cell Biology
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Primary antibodies for Grp78 (610978) and CPE (610758) were from BD (Transduction); GFAP (Z0334) and Ub (Z0458) from DAKO; Grp94 (SPA-850), PDI (SPA-891) and Hsp40 (Hdj1) (SPA-400) from Enzo; KDEL (PM059) and p62 (PM045) from MBL; APP (MAB348), ChAT (AB144P), Sec61β (07-205) and Ub (MAB1510) from MILLIPORE; HSP 60 (K-19) (sc-1722), NF-YA (sc-10779), Hsp90α (sc-8262) and Hsp70 (W27) (sc-24) from Santa Cruz; GFP (ab13970) and LacZ (ab9361) from abcam; Iba1 (019-19741) from WAKO; LacZ (200-4136) from Rockland; p62 (GP62-C) from PROGEN; Hsp40 (Hdj2) (MS-255-P) form Neomarkers; Lamin A/C (2032) from Cell Signaling.
7
Protein Expression Analysis of Cell Signaling
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After the cell culture experiments, whole-cell lysates were prepared for Arg-1, pSTAT6/STAT6, and pJNK/JNK, and nuclear extracts for NF-κB p65 subunit Western blots as previously described [24 (link)]. Protein concentrations of these extracts were measured by the Coomassie blue method [25 (link)]. SDS-polyacrylamide gel electrophoresis and Western blot analysis were carried out as previously described [26 (link)]. Antibodies to detect actin (sc-1615R) and Arg-1 (sc-18351) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while phospho-STAT6 (#56554), STAT6 (#9362), phospho-JNK (#9251), JNK (#9252), NF-κB p65 (#3034), and lamin A/C (#2032) antibodies as well as horseradish peroxidase (HRP)-linked goat anti-rabbit antibody (#7074) were all purchased from Cell Signaling Technology (Beverly, MA, USA).
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