Odyssey clx infrared scanner

NMuMG CRISPR cells lines expressing sfGFP-tagged exocyst subunits were grown to confluency in 100 mm cell culture dishes. Cells were washed with PBS and lysed in 1 ml of lysis buffer containing 20 mM HEPES; pH 7.4, 50 mM NaCl, 2 mM EDTA, and 0.1% Triton X-100, supplemented with cOmplete^TM mini EDTA-free protease inhibitor cocktails (Roche) and PhosStop (Roche). Cells were lysed in a rotator for 15 min at 4 °C and debris was removed by centrifugation at 16,000xg. For quantitative immunoprecipitation, protein amounts were measured using a BCA assay (ThermoFisher Scientific) and a small amount of lysates from each step (input, unbound, wash) were retained for analysis. Affinity purifications were performed with GFP-Trap_MA beads. Quantitative immunoblot analysis used secondary antibodies conjugated to IRDye^®680RD or 800CW (LI-COR) at 1:15,000 dilutions, imaging blots with Odyssey CLx Infrared Scanner (LI-COR). The uncropped immunoblot images are provided in Supplementary Fig 8.

Ten mg pancreatic tissue was ground in liquid nitrogen, and total protein was extracted by using RIPA lysis (CST, Danvers, MA, USA). Protein concentration was quantified using the BCA kit (TaKaRa, Dalian, China). HRP-conjugated goat anti-rabbit IgG H&L (ab6721) secondary antibodies were from Abcam (Abcam, San Francisco, CA, USA). The proteins were separated by SDS-PAGE and transferred to the PVDF membrane in the transfer buffer at 100 V for 2–3 h. The membrane was blocked for 1 hour at ambient room temperature in 10% nonfat milk, probed with antibodies against the above primary antibodies for 2 hours at 37°C, rinsed four times with PBTB, incubated 2 hours at 37°C in secondary antibodies, and washed extensively in PBS. Images were acquired on an Odyssey CLx infrared scanner (Li-Cor-Nebraska USA). Relative protein levels were calculated by using internal reference β-actin.

Cells were grown to 80% confluence in 6 cm dishes. After indicated treatments whole-cell lysates were prepared by sraping the cells in 150 µl lysis buffer (30 mM Hepes pH7.6, 1 mM MgCl₂, 130 mM NaCl, 0.5% Triton, 50 µM Mg132, EDTA-free protease inhibitor (Roche), and phosphatase inhibitor cocktail 2 (Sigma Aldrich)). 500U of benzonase (Millipore) was added per sample and incubated for 1 h on ice. For chromatin fractionation whole cell lysates were centrifuged for 15 min at 16100 × g and 4 °C. The supernatant containing nucleoplasmic RPB1 was collected. The pellet containing chromatin-bound RPB1 was washed twice with lysis buffer and re-suspended in 150 µl lysis buffer. Supernatant and pellet were diluted with 150 µl 2x SDS Page loading buffer (4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol) and separated on a 6% SDS PAGE gel. Proteins were transferred overnight at 4 °C at 35 V in 2x transfer buffer (25 mM TRIS, 190 mM Glycine) without methanol. Immobilon P PVDF membranes (Carl Roth) were blocked with 1.5% BSA in PBS and stained with antibodies as listed in Supplementary Table 1. Secondary antibodies were coupled to IRDyes (LiCor) and imaged with an Odyssey CLx infrared scanner (LiCor).

Following either sham or MCAO surgery, mice were anaesthetized with pentobarbital (100 mg/kg, i.p.) and transcardiacally perfused with ice-cold ACSF. Brains were instantly extracted and embedded in NEG50 (Richard-Allan Scientific, Fisher Scientific, Switzerland) and stored at −80°C. For immunostaining, 30 μm sections were cut using a cryostat (Hyrax 60, Carl Zeiss) and mounted onto glass slides (SuperFrost Plus, Thermo Scientific). The sections were fixated using 4% PFA for 30 min at room temperature, followed by antigen retrieval in a PickCell 2100-Retriever in 10 mM citrate buffer pH 6.0, 0.05% Tween 20 for 20 min. Sections were then permeabilized (0.2% Triton X-100 in 10 mM Tris pH 7.4, 150 mM NaCl, 10% NGS) and incubated with primary antibody diluted in TBST (10 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20) containing 10% NGS overnight at 4°C. After washing five times for 5 min with TBST, the sections were incubated with secondary antibodies (Alexa Fluor Plus 800) for 1 h at room temperature. After washing with TBST, sections were air dried in the dark and scanned with an Odyssey CLx infrared scanner (Licor) at a resolution of 21 μm (Figures 4A, 6A) and 42 mm (Figure 3C).

Ten milligram colon tissues were ground in liquid nitrogen and total protein was extracted by using RIPA lysis (CST, Danvers, MA, USA). Protein concentration quantified using the BCA kit (TaKaRa, Dalian, China). HRP-conjugated goat anti-rabbit IgG H&L (ab6721) secondary antibodies were from Abcam (Abcam, San Francisco, CA, USA). The proteins were separated by SDS-PAGE and transferred to the PVDF membrane in the transfer buffer at 100 V for 2–3 h. The membrane was blocked for 1 h at ambient room temperature in 10% non-fat milk, and probed with antibodies against the above primary antibodies for 2 h at 37°C, rinsed four times with PBTB, incubated 2 h at 37°C in secondary antibodies, washed extensively in PBS. Images were acquired on an Odyssey CLx infrared scanner (Li-Cor- Nebraska USA). Relative protein levels were calculated by using internal reference β-actin.