RPL1 | 50S ribosomal protein L1

Ribosomal proteins, translation activity and total protein contents in the ?sigBCDE and control (CS) strains in standard growth conditions. (A) Total proteins were isolated, separated by SDS-PAGE and then the Rps1 protein of the 30S ribosomal subunit and the Rpl1 protein of the 50S ribosomal subunit were detected with western blotting. As a loading control the amount of the ? subunit of ATPase was detected as well. Original Western blots are shown in Supplementary Fig. S1. (B) Cells were labelled with radioactive 35S-methionine for 10 or 30?min, as indicated, in standard growth conditions, and total proteins were isolated after addition of excess of cold methionine. Proteins were separated by SDS-PAGE, transferred to the membrane and visualized by autoradiography. (C) Total proteins were isolated from the same amount of cells and the protein content was detected with a BioRad DC protein assay kit. Three independent biological replicates were measured, Student’s t-test P?=?0.905.

Ribosome profiles of the control (CS) and ?sigBCDE strains. The ribosomes of the CS (A,B) and ?sigBCDE (C,D) strains were fractionated with a 5–35% sucrose density gradient centrifugation, nineteen fractions were collected and either RNA or ribosomal proteins Rps1 and Rpl1 were detected from each fraction. RNA was isolated from the 300??L sample of fraction, dissolved in 20??L of water, and 15??L of isolated RNA from each fraction of CS (A) or ?sigBCDE (C) was loaded on a 1.2% agarose gel and stained with ethidium bromide. The DNA molecular marker was included to demonstrate separation of fragments. For western blots, a 24??L sample of a fraction was solubilized and proteins were separated on SDS-PAGE and Rps1 and Rpl1 proteins were detected by specific antibodies by western blotting in the control (B) and ?sigBCDE (D) strains.

Ribosome profiles of the control (CS) and ?sigBCDE strains. The ribosomes of the CS (A,B) and ?sigBCDE (C,D) strains were fractionated with a 5–35% sucrose density gradient centrifugation, nineteen fractions were collected and either RNA or ribosomal proteins Rps1 and Rpl1 were detected from each fraction. RNA was isolated from the 300??L sample of fraction, dissolved in 20??L of water, and 15??L of isolated RNA from each fraction of CS (A) or ?sigBCDE (C) was loaded on a 1.2% agarose gel and stained with ethidium bromide. The DNA molecular marker was included to demonstrate separation of fragments. For western blots, a 24??L sample of a fraction was solubilized and proteins were separated on SDS-PAGE and Rps1 and Rpl1 proteins were detected by specific antibodies by western blotting in the control (B) and ?sigBCDE (D) strains.