In vivo single-cell ribosome profiling reveals cell-type-specific translational programs during aging. Duré et al.

Description

Original images of Western blot images: Replicate 1: used in Figure 4I Sample loading scheme: Lane 1 = Young Sca-1 pos; Lane 2 = Old Sca-1 pos; Lane 3 = Young Sca-1 neg; Lane 4 = Old Sca-1 neg; Lane 5 = Marker; Lane 6 = in vitro keratinocytes ctrl; Lane 7 = in vitrokeratinocytes STRESS "Rep1_cJun_Chemi.tif" shows c-Jun protein bands and is used in the manuscript in Figure 4I. Image where the marker and membrane label "2", standing for "gel 2" is visible can be found in "Rep1_cJun_Chemi+Marker.jpg" "Rep1_vinculin_Chemi.tif" is used in the manuscript in Figure 4I. Two membranes were imaged at the same time. Therefore a second images is provided, showing the gel/membrane numbering (pencil label "gel 1", "gel2"). The WB image used in Figure 4I originates from gel 2 (lower bands.) Replicate 2: used for quantification in Figure S4L: Loading scheme: Lane 1 = Young Sca-1 pos; Lane 2 = Old Sca-1 pos; Lane 3 = Young Sca-1 neg; Lane 4 = Old Sca-1 neg; Lane 5 = Marker; Lane 6 = in vitro keratinocytes ctrl; Lane 7 = in vitro keratinocytes STRESS "Rep2_cJun_Chemi.tif" shows c-Jun protein bands. "Rep2_Vinc_Chemi.tif" shows Vinculin protein bands. "Rep2_cJun_Chemi+Marker.jpg" shows cJun protein bands with markers. "Rep2_Vinc_Chemi+Marker.jpg" shows Vinculin protein bands with markers. Only tif images were used for quantification.

Steps to reproduce

Epidermal cells were isolated from young (2 month old) and aged (26 month old) animals as described above. The cell suspension was stained with Ly-6A/E (Sca-1) Monoclonal Antibody, FITC (1:100, Thermo Fisher Scientific, 11-5981-82) for 30 minutes at 4°C, followed by a wash with FACS buffer and resuspension in FACS buffer supplemented with DAPI (0.5 ng/ml). Cells were sorted on a BD FACSAria™ III Cell Sorter using a 70 μm nozzle. Cells were collected in E-low medium and centrifuged at 400 g for 15 minutes. The cell pellet was lyzed in RIPA buffer (Sigma-Aldrich, R0278), supplemented with Protease Inhibitor Cocktail (50x, Promega, G6521), Phosphatase inhibitor cocktail 2 (100x, Sigma, P5726-1ML) and Phosphatase inhibitor cocktail 3 (200x, Sigma, P0044-1ML) and incubated on ice for 10 minutes. The cell lysate was centrifuged at full speed for 10 minutes at 4°C and supernatant was collected. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Protein lysates were mixed with 4x sample loading buffer (0.2 M Tris-HCL, 0.4 M DTT, 8% SDS (w/v), 6 mM bromphenol blue, 4.3 M glycerol) and heated to 95°C for 10 minutes, then stored at -80°C. Protein samples were re-heated to 95°C for 10 minutes and loaded on a 4-12% NuPage 12-well Bis-Tris Gel (Invitrogen, 7001691) using NuPAGE MOPS SDS Running Buffer (20X, Invitrogen, NP0001). Proteins were subsequently transferred to a nitrocellulose membrane (GE Healthcare) by tank transfer at 4°C, 30V for 1.5 hours. Membranes were blocked in 5% skimmed milk. Primary antibodies were incubated overnight at 4°C and secondary antibodies for 2 hours at 4 °C. Western blots were developed with freshly mixed ECL solutions (GE Healthcare). The following antibodies were used: c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, 9165), Phospho-4E-BP1 (Thr37/46) (Cell Signaling, 2855), Anti-Vinculin (Abcam, ab129002). Western blot bands were quantified using ImageJ.

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• University of Zurich

  Zurich, Zurich

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