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Figure 2. Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES- interacting proteins PTB, Ebp1 and <t>Gemin5.</t> This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).
Gemin5, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Figure 2. Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES- interacting proteins PTB, Ebp1 and Gemin5. This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).

Journal: Scientific reports

Article Title: Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE.

doi: 10.1038/s41598-018-23845-x

Figure Lengend Snippet: Figure 2. Preparation of cellular fractions. (A) Schematic of the procedure used to prepare cellular fractions S30, S100, R, RSW, and F from HEK293 cells. S30 extract is the total lysate obtained from cells. S30 ultracentrifugation yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (R) (pellet). To prepare the fraction containing ribosomes free from associated factors (RSW), the ribosomal pellet was dissolved in high-salt buffer, loaded in a discontinuous sucrose gradient and ultracentrifuged. The supernatant of the ultracentrifugation yielded the F fraction. (B) HEK293 fractions corresponding to S30, S100, F (100 µg of total protein), ribosomes (R) and salt-washed ribosomes (RSW) (30 µg) were analyzed by Western blot on the same membrane to detect the presence of RACK1 (40 S subunit), the 60S ribosomal proteins P0 and P1/P2, the elongation factor eEF2, the initiation factors eIF4G, eIF4B, eIF4E, and eIF2α, and the IRES- interacting proteins PTB, Ebp1 and Gemin5. This figure shows horizontal slices of the WB carried out for each factor. Images of the un-cropped WB film obtained for each factor are shown in Supplementary Fig. S7).

Article Snippet: Commercial antibodies were used to detect eIF4E (Transduction laboratories), RACK1, eIF2α, eIF4G (Santa Cruz), eEF2 (Cell Signaling), eIF4B, and Gemin5 (Novus).

Techniques: Western Blot, Membrane