Irdye 800 goat anti mouse igg

SDS-PAGE and Western blot was performed on tumor cell lysates by the methods described previously21 (link). For immunoblotting, cell lysates (n = 4) (10 μg) resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes were probed with primary antibodies [mouse anti-human (1:1000)], TOP2A (Cell Signaling Technology, Danvers, MA, USA), PYCR2 and PPL (Santa Cruz Biotechnology, Dallas, USA) for 1 h in TTBS [50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20] followed by incubation with corresponding secondary antibodies; IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG (1:15000, LI-COR Biosciences), for 1 h at room temperature in TTBS. Immunoblots were imaged using the CLx Odyssey Infrared Imaging System, (v3.0, LI-COR Biosciences, Nebraska USA). Loading controls were obtained by staining the membrane with Deep Purple Total Protein stain as previously described15 (link)16 (link). Semi-quantitative densitometric analysis was performed on all blots (3 biological replicates) to determine the level of protein expression using ImageStudio v5, with mean pixel intensity of the protein of interest normalised to the background.

Histone samples were exposed to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (14%), transferred to a PVDF membrane, blocked with Odyssey blocking buffer (LI-COR Biosciences) or 5% non-fat milk for 1 hr, and incubated with primary antibodies overnight at 4°C as well as with secondary antibodies conjugated to fluorescence (IRDye 680 goat anti-rabbit IgG or IRDye 800 goat anti-mouse IgG, Li-Cor) for 1 hr at room temperature. Bands were visualized in an Odyssey Infrared Imaging System (Li-Cor). Coomassie staining or histone H3/histone H4 immunoblotting were used as loading controls. H1 protein content was quantified from Coomassie staining of histone extracts using ImageJ software. H1 variants can be visualized in three consecutive bands (35–32 kDa, corresponding to H1.3 + H1.4 + H1.5, H1.2, and H1.0, respectively), as indicated in Figure 5—figure supplement 1A, C. H1X cannot be quantified from Coomassie staining. The relative intensity of each H1 band was corrected by H4 band (loading control) and expressed as a percentage of total H1 content.

PDAC cells were serum starved for 48 h and treated as described in the text. In accordance with the manufacturer’s instructions, subcellular fractions were prepared using Thermo Fisher Scientific’s NE-PER Nuclear and Cytoplasmic Extraction reagents. Bicinchoninic protein assay kits (Pierce) were used to measure protein concentrations. We loaded equal amounts of protein in each lane, separated the proteins on a 4–12% Bis-Tris NuPAGE gel (Invitrogen) and transferred the proteins to PVDF membranes. The following primary antibodies and infrared secondary antibodies were used to probe the membranes: IRDye 700 goat anti-rabbit IgG and IRDye 800 goat anti-mouse IgG (LI-COR Biosciences). LI-COR Biosciences’ Image Studio software (LI-COR) was used to quantify protein bands using infrared signals detected by the Odyssey CLx infrared imaging system.

Western blotting was performed as described previously [8 (link)]. Briefly, normalized concentration protein was loaded on a 10% SDS–polyacrylamide gel and separated by electrophoresis. Then, the protein was transferred from the gel to a polyvinylidene difluoride (PVDF) membrane. After blocking with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), the membrane was probed using rabbit anti-Dicer (1:2000, Novus Biologicals, Littleton, CO, USA), rabbit anti-PCNA (1:1000, Cell signaling Technology), rabbit anti-phospho-Histone H3 (1:1000, Cell Signaling Technology), and mouse anti-β Actin (1:5000, Novus Biologicals, Lincoln, NE, USA) at 4 °C overnight. After washing with 1 × TBST, the membrane was incubated for 1 h at room temperature with IRDye 680 goat anti-rabbit IgG or IRDye 800 goat anti-mouse IgG (1:10,000, LI-COR Biosciences). The probed blot was scanned using an Odyssey infrared imager.

Western blotting was performed as described before (Ruan et al., 2018a (link)). Briefly, proteins (normalized for concentration) were resolved on 10% SDS–polyacrylamide gels and transferred onto Odyssey^® nitrocellulose membranes (LI-COR Biosciences). The membranes were blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, United States) and probed with rabbit anti-Rab27a (1:1000; Cell Signaling), rabbit anti-Rab27b (1:1000; Millipore), rabbit anti-Tri-methyl-Histone H3 (Lys4) (1:1000; Cell Signaling), mouse anti-TBP (1:1000, Proteintech), and mouse anti-β-actin (1:5000, Novus Biologicals) at 4°C overnight. After washing with 1 × TBST, the membrane was incubated for 1 h at room temperature with IRDye 680 goat anti-rabbit IgG and IRDye 800 goat anti-mouse IgG (1:10,000, LI-COR Biosciences). The probed blot was scanned using an Odyssey infrared imager (LI-COR Biosciences).

Cells were lysed in RIPA buffer (Sigma) with phosphatase and protease inhibitors (EMD Millipore). Protein content was measured by bicinchoninic acid (BCA) assay and used to normalize samples to the lowest concentration. Lysates were heated to 95°C and run on a 4-12% Bis-Tris gel (Life Technologies) and transferred to a nitrocellulose membrane. Membranes were blocked in Odyssey blocking buffer (Li-Cor) and incubated with primary antibody at 4°C overnight (Table S2). All membrane wash steps were performed using Tris-buffered saline with 0.1% Tween-20. Membranes were incubated with secondary antibody, IRDye 800 goat anti-mouse IgG and IRDye 700 goat anti-rabbit IgG (Li-Cor) at a 1:10,000 dilution in blocking buffer for 1 h at room temperature. Blot fluorescence was visualized using an Odyssey CLx system (Li-Cor) and quantified with the built-in gel analyzer tool in ImageJ.