Protein content of lysates was determined using the bicinchoninic acid assay. Samples were separated by SDS PAGE with precast gels and subsequently the proteins were transferred to nitrocellulose membrane with a semi dry blotting system as described. Membranes were blocked with TBST containing 0. 1% Tween 20 and 5% milk powder for 1 h at RT fol lowed selleckbio by incubation with primary antibodies, 1,500, rabbit polyclonal anti EpoR 1,1000, mouse monoclonal anti GAPDH 1,10,000, mouse monoclonal anti b actin 1,10,000, rabbit polyclonal anti HIF 1a 1,500, all Santa Cruz overnight at 4 C in blocking buf fer. Afterwards blots were rinsed 3 times with TBST and incubated with fluorescent dye labelled secondary antibodies. As a molecular weight marker, the pre stained peqGOLD marker IV was used.
Visualization and quantification were performed with Odyssey Infrared Imaging System. Immunoblots were scanned at a wavelength of 700 nm for Alexa Fluor 680 labeled antibodies and at a wave length of 800 nm for IRDye 800CW labeled antibodies, respectively using Odyssee software version 1. 2. Expres sion of b actin or GAPDH were used for normalization. Values were normalized and thereby relative expression levels of the target proteins were determined. Nuclear encoded mitochondrial proteins synthesized in the cytosol are targeted to the mitochondria by one of two types of targeting signals, a hydrophobic prese quence and or a cryptic internal sequence. The MLS directs the precursor protein to the translo case of the outer membrane where transloca tion begins.
In addition, the MLS affects the precursor import efficiency as determined by the length of signal peptide and encodes the submitochondrial localiza tion of mitochondrial proteins after mitochondrial pro cessing, as exemplified by the presence of a cleavable or non cleavable stop transfer signal. Redistribution after mitochondrial processing can also be affected by protein folding, even though most precursor transloca tion requires unfolding. Of the two reported examples of protein folding affecting mitochondrial import, the propeller domain of PP2A Bb2 subunit arrests the import process and becomes on OMM protein whereas rapid folding of yeast fumarase during the import favors the retrograde movement for a cytosolic localization.
Interestingly, there are only a handful of proteins that distribute between the mitochondria and cytosol in a constitutive manner, fumarase being the most studied example. It has been demonstrated that fumarase has a 30% Brefeldin_A 70% mitochondria cytosol isopro tein distribution and this dual localization occurs after mitochondrial processing. The PINK1 gene encodes a kinase protein that con tains an N terminal MLS and mutations in PINK1 are linked to a recessive form of Parkinsons disease.