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. 2015 Dec 9;4:e07624. doi: 10.7554/eLife.07624

Figure 2. Localization of the DrCNGK channel.

Figure 2.

(A) Western blot of membrane proteins (15 µg) from CHOK1 cells transfected with cDNA encoding either DrCNGK with a C-terminal HA-tag alone (lane C) or with both, a C-terminal HA-tag and an N-terminal flag-tag (N/C). Apparent molecular weight Mw is indicated on the left. (B) Characterization of anti-DrCNGK antibodies. Left: Western blot of membrane proteins (10 µg) from HEK293 cells transfected with cDNA encoding DrCNGK (Tr) and wild-type cells (wt). Right: Western blot of membrane proteins (15 µg) from zebrafish testis. (C) Western blot of membrane proteins (15 µg) from different zebrafish tissues. (D) Upper panel: Scheme of a testis cross-section. GC, germinal compartment; IC, intertubular compartment; SER, Sertoli cells; SGA, primary spermatogonia; SGB, secondary spermatogonia; SC, spermatocytes; ST, spermatids; scheme according to (Nobrega et al., 2009). Lower panel: Staining with anti-repeat1 antibody (red, left) and superposition (right) of the immunohistochemical image with a bright-field image of an in situ hybridization using an anti-DrCNGK-specific RNA probe (arrows). Bar represents 50 µm. (E) Staining of zebrafish sperm with anti-repeat1 (upper left panel) and anti-repeat3 antibody (lower left panel). Bars represent 10 µm. The respective bright-field images are shown (upper and lower right panels). (F) Western blot of equal amounts of total membrane proteins (15 µg) from purified heads and purified flagella.

DOI: http://dx.doi.org/10.7554/eLife.07624.007

Figure 2—source data 1. Indicators of merit for the mass spectrometric results of a testis preparation.
a m indicates oxidized methionine, b△M [ppm] relative mass error, c XCorr, △Score, and △Cn indicate results based on searches with the sequest algorithm in Proteome Discoverer; d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event.
DOI: http://dx.doi.org/10.7554/eLife.07624.008
elife-07624-fig2-data1.xlsx (14.6KB, xlsx)
DOI: 10.7554/eLife.07624.008
Figure 2—source data 2. Indicators of merit for the mass spectrometric results of different sperm preparations: whole sperm, head and flagella. .
a m indicates oxidized methionine, b△M [ppm] for all peptides is below ± 2.5, c XCorr indicates results based on searches with the sequest algorithm; Amanda indicates results based on searches with the MS Amanda algorithm, d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event; △Score and △Cn are not indicated separately since the scores for all peptides are 1 and 0, respectively.
DOI: http://dx.doi.org/10.7554/eLife.07624.009
elife-07624-fig2-data2.xlsx (22KB, xlsx)
DOI: 10.7554/eLife.07624.009