Figure 1
From: The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function
Detection of RPE65 palmitoylation. (A,B) Analysis of palmitoylation of RPE65 from RPE microsomal extracts and HEK293F-expressed lysates was performed by acyl-RAC and ABE assays. Rhodopsin and CRALBP were used as positive and negative control proteins, respectively. Samples were treated with a final concentration of 0.5 M hydroxylamine (HAM) or 0.5 M NaCl (control) and then palmitoylated proteins were enriched using beads (thiopropyl-sepharose for acyl-RAC and streptavidin-agarose for ABE) were eluted using 2.5% β-mercaptoethanol in SDS-PAGE sample buffer. Equal amounts (~20 µg) of total (indicated as “input”) and eluted protein from control (indicated as “−”) and HAM-treated (indicated as “+”) samples were separated by SDS-PAGE followed by immunoblotting with primary antibody to RPE65. Results are representative of three independent experiments. (C) The bar graph indicates normalized palmitoylation percentage averaged from three different experiments of Acyl-RAC and ABE assays. Error bars represents standard deviation. (D) Following Acyl-RAC, eluted proteins from control and HAM-treated samples separated by SDS-PAGE were stained using blazin blue protein gel stain. Asterisk shows enriched RPE65 band at ~65 kDa position in the HAM-treated sample. Box shows the identified peptides of RPE65 in the HAM-treated sample using in-gel trypsin digestion and mass spectrometry.
