Figure 1: Identification of pTH2A in mouse spermatozoa.
From: Identification of a variant-specific phosphorylation of TH2A during spermiogenesis
(a) An experimental scheme to prepare nuclear fractions from spermatozoa. Proteins are biochemically divided into three fractions (S1, S2, and Ppt). Silver staining and western blot analyses of each fraction are as indicated. The red box indicates the area to be digested and subjected to LC-MS analysis. (b) Representative MS/MS spectrum of pTH2A peptides (upper panel). The y-axis indicates intensity of MS/MS spectra, and the x-axis indicates m/z. Phosphorylated threonine is highlighted in red, and the identification number for each peptide is summarized (lower panel). (c) Alignment of amino acid sequences between mouse H2A, mouse TH2A, and human TH2A. Black arrow, start of the C-terminal tails; red box, the phosphorylated threonine in mouse TH2A; green line, epitope sequence for generation of the anti-pTH2A antibody; Roman numeral, number of amino acid from the N-terminus. (d) Western blot analysis of sperm S2 fraction treated or untreated with lambda protein phosphatase (λPPase). Experimental procedure (left panel) and representative images of western blot analysis (right panel) are indicated. (e) Peptide blocking assay. Indicated peptides were added to the diluted primary antibody during the reaction. (f) Western blot analysis of pTH2A (upper panel) and TH2A (lower panel). Protein extracts of HEK293T cells overexpressing FLAG-tagged H2A (F-H2A) and -TH2A (F-TH2A), whole testicular cells, germ stem cells (GS cells), and spermatozoa were analyzed. Number of biological replicates of each experiment is shown as n.
