Rabbit anti lamin a c

Cells were lysed with Cell Lysis buffer (1X) supplemented with protease inhibitor cocktail (Cell Signaling, USA). Total cell lysates (5–10 μg) were resolved on 4–12% Bis-Tris Nupage gels (Invitrogen, USA) and transferred onto PVDF membranes (Millipore) and developed with antibodies for GAPDH, NF-κB p65, Phospho-NF-κB p65, and anti-Rabbit IgG HRP-linked antibody using SuperSignal West Femto Maximum Sensitivity kit (Thermo Scientific), Data quantification was done using Image J software (NIH).
Alternatively, MOC1 and SUM149 cells were plated at 2 × 10 6 cells in T75 flasks and allowed to adhere overnight. Cells were pre-treated with 0.1% DMSO, 20 μM HP163 for 30 min and then treated with 0.1% DMSO, 100 μM Kyn or 1 nM TCDD for 1 hour. Nuclear and cytoplasmic cell extracts were prepared using a Nuclear Extract Kit (Thermo Scientific) as per the manufacturer’s instructions. Protein concentration was quantified using the Protein Assay Reagent (Bio-Rad). Protein (30 μg) was resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes were probed with mouse anti-AHR (Pierce, cat #MA1-514), rabbit anti-Lamin-A/C (Cell Signaling, cat #2032) and mouse anti-α-tubulin (EMD Millipore, cat #CP06). Immuno-reactive bands were detected using HRP-conjugated secondary antibodies (goat anti-rabbit, Bio-Rad; goat anti-mouse Pierce) and ECL substrate.

In these experimental procedures we used the following antibodies: rabbit polyclonal antibody against COMMD1 (11938-1-AP, Proteintech Group, USA), mouse anti-β-Actin (A5441, Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands), rabbit anti-Tubulin (AB4047, Abcam, Cambridge, UK), rabbit anti-Lamin A/C (2032, Cell Signaling Technology Europe, B.V., Leiden, the Netherlands), rabbit anti-p65 (4764, Cell Signaling Technology, Europe, B.V.), rabbit anti-IκBα (sc-371, Santa Cruz Biotechnology Inc., Heidelberg, Germany), rabbit anti-Cd68 (#137002, Biolegio, Nijmegen, the Netherlands) rabbit anti-F4/80 (#101201, Biolegio, Nijmegen, the Netherlands), goat anti-rabbit IgG (H + L)-HRP Conjugate (170-6515, Bio-Rad Laboratories BV, Veenendaal, the Netherlands), goat anti-mouse IgG (H + L)-HRP Conjugate (170-6516, Bio-Rad Laboratories BV).

A Nu-Per kit (Thermo Fisher, Scientific, Grand Island, NY) was used to perform subcellular fractionation of cell pellets. Lysates were then quantitated by Pierce BCA Protein Assay Kit (Thermo Fisher) and loaded equally onto SDS-PAGE gels. Proteins were electrotransferred onto Immobilon-P membranes (EMD Millipore, Billerica, MA) and incubated with mouse anti-HuR (Santa Cruz, Dallas, TX, USA), rabbit anti-Lamin A/C (Cell Signaling, Danvers, MA, USA), anti-DR4 (Thermo Fisher) or mouse anti-α-tubulin (Life Technologies) primary antibodies for 12 h in Odyssey blocking buffer (Licor, Lincoln, NE). The corresponding secondary antibodies were used at 1:10,000 dilutions (Santa Cruz). Immunoblots were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences, model #9120). Densitometric quantification was performed using Odyssey Infrared Imaging System software to verify the amount of protein on each immunoblot. The numbers under sample bands indicate the densitometric quantification values, first normalized to their respective loading control and expressed as fold-change compared to their respective control sample.

Cells were fixed in 4%PFA/PBS for 15 minutes, permeabilized with 0.1% Triton-X-100/PBS and then blocked in 5% FBS supplemented with 0.1% BSA for 1 h. For BrdU, cells were treated with an additional incubation in 2N HCl for 1 h at room temperature after the permeabilization. Primary antibodies were incubated overnight at 4°C in blocking solution at the following concentrations: mouse anti-Satb2 (SATBA4B10, SantaCruz) 1:300; mouse anti-alpha Tubulin (DM1A) 1:500; mouse anti-Lamin B1 (B-10, SantaCruz) 1:100; rabbit anti-Lamin A/C (Cell Signaling Technology) 1:100; rabbit anti-BrdU (Abcam) 1:50. Cells were imaged on a Nikon AR-1, a Leica Sp8, or a Zeiss Axiovert 200M microscope. Images were processed using the Fiji distribution of ImageJ and Adobe Photoshop CC 2018 (Schindelin et al., 2012 (link)).