Bleomycin was purchased from Thermo Fisher Scientific. The following antibodies were used in Western blot and immunofluorescent staining. RGC32 polyclonal antibody was produced by Proteintech Group, Inc. (Chicago, IL) (16 (link)). Collagen type I alpha 1 (COL1A1) (D-13) and Lamin B (C-20) were obtained from Santa Cruz Biotechnology. Inducible nitric oxide synthase (iNOS) (4E5), Arginase (4E6) and CD3 were purchased from Abcam. IL-1β (3A6), NF-κB p65 (D14E12), Phospho-NF-κB p65 (Ser536), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) (44D4) and Phospho-IκB (Ser32) were from Cell Signaling Technology. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Proteintech. F4/80 (BM8) was from BioLegend. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc.). The secondary antibodies were from Cell Signaling Technology. M-CSF and IFNγ were purchased from R&D Systems. M-CSF was used at 10 ng/mL, and IFNγ was used at 100 ng/mL respectively. LPS was obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and used at 100 ng/mL.
Lamin b c 20
Manufactured by Santa Cruz Biotechnology
Sourced in Germany, United Kingdom, United States
Lamin B (C-20) is a protein used in research applications. It serves as a structural component of the cell nucleus. This product is intended for laboratory use only.
Automatically generated - may contain errors
Lab products found in correlation
Bleomycin, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
M csf, by R&D Systems (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
F4 80 bm8, by BioLegend (1 mentions)
F4 80 bm8 antibody, by BioLegend (1 mentions)
Gapdh antibody, by Proteintech (1 mentions)
Raptor 24c12, by Cell Signaling Technology (1 mentions)
Phosphotyrosine 4g10, by Merck Group (1 mentions)
Odyssey system, by LI COR (1 mentions)
P akt1 2 3 ser473, by Santa Cruz Biotechnology (1 mentions)
Anti foxo3a 75d8, by Cell Signaling Technology (1 mentions)
P44 42 mapk, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
β actin ac 15, by Santa Cruz Biotechnology (1 mentions)
P foxo3a, by Cell Signaling Technology (1 mentions)
Ha 3f10, by Roche (1 mentions)
Chk1 g 4, by Santa Cruz Biotechnology (1 mentions)
11 protocols using lamin b c 20
1
Macrophage Polarization Assay Protocol
2
Investigating Bleomycin-Induced Inflammatory Pathways
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Bleomycin was purchased from ThermoFisher Scientific. The following antibodies were used in Western blot and immunofluorescent staining. ADAR1 (D-8) and collagen type Ia1 (COL1a1) (D-13) and Lamin B (C-20) were obtained from Santa Cruz Biotechnology. iNOS (4E5) were purchased from Abcam. IL-1 b (3A6), NF-κB p65 (D14E12), phospho- NF-κB p65 (Ser536) were from Cell Signaling Technology. GAPDH antibody was from Proteintech, and F4/80 (BM8) antibody was from BioLegend. Nuclei were stained with DAPI (Vector Laboratories). The secondary antibodies were from Cell Signaling Technology. M-CSF and IFNγ were purchased from R&D Systems. M-CSF was used at 10 ng/ml, and IFNγ was used at 100 ng/ml. LPS was obtained from Sigma-Aldrich (St. Louis, MO) and used at 100 ng/ml.
3
Immunoblotting Techniques for Phosphoproteins
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Standard immunoblotting techniques were used with the following antibodies: phosphotyrosine (4G10; EMD Millipore), Lamin B (C20; Santa Cruz Biotechnology, Inc.), phosphoAkt Thr308 (244F9, 4056), phosphoAkt Ser473 (9271), Akt1 (2H10, 2967), mTOR (7C10), Rictor (53A2), and Raptor (24C12; Cell Signaling Technology). Blots were quantified using fluorescent secondary antibodies and an Odyssey system (Licor).
4
Western Blot Protein Detection
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Total, cytosolic and nuclear proteins were extracted and counted as previously described [23 (link)]. Lysates were separated on SDS-PAGE gel transferred to nitrocellulose membranes and proteins were detected with specific polyclonal (p) or monoclonal (m) antibodies (Abs), recognized by IRDye secondary Abs (LI-COR Corporate, Milan, Italy). The Abs employed were anti-FoxO3a (75D8, #2497), p-FoxO3a (Ser253; #13129), p-FoxO3a (Ser294; #5538), p44/42 MAPK (#9102), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101) (all from Cell Signaling Technology Europe, B.V., Leiden, The Netherlands). MDM2 (IF2; #33-7100) (ThermoFisher Scientific), AKT 1/2/3 (H136; sc-8312), p-AKT 1/2/3 (Ser473) (sc-7985), p21 (sc-71811), p27 (sc-53871), p53 (sc-126), CD1 (sc-718), β-Actin (AC-15; sc-69879), GAPDH (FL-335; sc-25778) and Lamin B (C-20; sc-6216) all from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. Images were acquired with the Odyssey FC Imaging System (LI-COR Corporate). For each Western blot figure, the whole blots showing all the bands with all molecular weight markers, as well as the densitometry readings/intensity ratio of each band (analyzed using Image J software, NIH, USA) are collected in the Supplemental Materials as a separate file named “Original blots and densitometry”.
5
Antibody Panel for DNA Damage Response
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The following antibodies were used: PCNA (FL261 and PC10; Santa Cruz Biotechnology), HA (3F10; Roche), Polη [15 (link)], REV1 [15 (link)], LaminB (C20; Santa Cruz Biotechnology), RAD18 (rabbit polyclonal), phospho-CHK1 (Ser345) (133D3; Cell Signaling), CHK1 (G4; Santa Cruz Biotechnology), actin (I19; Santacruz), γH2AX (Millipore), Ub (P4D1; Santa Cruz Biotechnology), and FLAG (M2; Sigma).
6
Antibody Reagents for Molecular Analysis
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Mibolerone (Mb) was from Sigma Aldrich (St. Louis, MO, USA). The antibodies against β-Actin (AC-15), BAD (C-7), BAX (B-9), Bcl-2 (C-2), BID (FL-195), GAPDH (FL-335), and Lamin B (C-20) were from Santa Cruz Biotechnology (Bolivia); OXPHOS and VDAC1 were from Abcam (Cambridge, UK); and AR (D6F11) was from Cell Signaling (Boston, MA, USA).
7
Protein Quantification by Western Blot
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Following stimulation, total cell extracts were collected at the indicated times. To detect the protein levels, cell lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Analyses were performed using Amersham ECL Prime Western Blotting Detection Reagent (RPN2232) (GE Healthcare Life Sciences, Chicago, IL), and the membranes were scanned using Image Quant LAS4010 (GE Healthcare Life Sciences). We used primary antibodies for ERK (#9102), phospho-ERK (#4370), AKT (#9272), phospho-AKT (#9271), p21 waf/cipl (12p1) (#2947) (Cell Signaling Technology), Lamin B (C-20) (#sc-6216) (Santa Cruz, Dallas, TX), DPT (AF4629; R&D systems, Minneapolis, MN), and β-actin (#ab6276; Abcam).
8
Protein Expression Analysis by Western Blot
9
Immunoblotting of Epithelial-Mesenchymal Transition Markers
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Antibodies recognizing E-cadherin (24E10) (3195), Snail (C15D3) (3879), ROCK1 (C8F7) (4035), ROCK2 (D1B1) (9029), cofilin (D3F9) (5175), phospho-cofilin (Ser3) (77G2) (3313), phospho-β-catenin (S33/37/41) (9561), phospho-GSK-3β (Ser9) (D85E12) (5558), GSK-3β (D5C5Z) (12456), Axin2 (76G6) (2151), Sox-9 (D8G8H) (82630) ZO-1 (D6L1E) (13663), and DKK-1 (D5V6L) (48367) were obtained from Cell Signaling Technology (Danvers, MA). Additional antibodies used were E-cadherin (BD Transduction Laboratories, 610181), β-catenin (BD Transduction Laboratories, 610153), c-myc (Abcam, Cambridge, United Kingdom; ab32072), GPCR GPR49 (Lgr5) (Abcam, ab75850), GAPDH (GeneTex, Irvine, CA; GTX627408), Lamin B (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-6216), and β-actin (C4) (Santa Cruz Biotechnology, sc-47778). Secondary HRP-conjugated antibodies used were rabbit immunoglobulin G HRP-linked (GE Healthcare, Chicago, IL; GENA934) and mouse immunoglobulin G HRP-linked (GE Healthcare, GENA931).
10
Nuclear extract and Western blot analysis of NR4A1 in MA-10 Leydig cells
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Nuclear extracts and western blots were performed as described previously (Martin et al. 2008 (link), Abdou et al. 2014) (link). For each experiment, MA-10 Leydig cells were cultured in serum-free media in the presence of either vehicle, Fsk (10 μM) alone, Fsk + dantrolene (150 µM), Fsk + W7 (40 µM) for 1 h before nuclear extracts preparation. Each experiment was performed at least 3 times. The antisera used for detection of NR4A1 (M-210), LAMIN B (C-20) and CREB (H-74) were purchased from Santa Cruz Biotechnology.
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