Fig. 2: piRNA loading facilitates MIWI dissociation with TDRKH. | Nature Communications

Fig. 2: piRNA loading facilitates MIWI dissociation with TDRKH.

From: piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis

Fig. 2

a, b Co-IP assay of the association of MIWI and TDRKH in mouse testes from 18 dpp wildtype, MiwiYY/YY, and MiwiYK/YK mice. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting]. c RNA co-IP assay of MIWI-interacting piRNAs in anti-MIWI (lane 1) and anti-TDRKH IP pellets (lane 2) from adult wildtype mouse testes, with anti-MIWI IB as a loading reference (bottom). d Anti-MIWIunloaded preferably pulled down piRNA-unloaded MIWI (left) and TDRKH (right) in adult mouse testicular lysate. Left, RNA co-IP assays using anti-MIWIunloaded and control anti-MIWI antibodies in adult wildtype mouse testicular lysate, with anti-MIWI IB as loading references. Right, co-IP assay of the association of MIWI and TDRKH using anti-MIWIunloaded (lane 3) and control anti-MIWI antibodies (lane 4) in adult wildtype mouse testicular lysate, with testicular lysate (lane 1) and IgG IP (lane 2) serving as positive and negative controls, respectively. Quantification of blot intensity of TDRKH is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting]. e RNase A treatment enhanced the MIWI-TDRKH interaction in wild-type mouse testes. Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from RNase A-untreated (lane 4) is set as 1.0 after normalization with MIWI blotting]. f Transfection of piRNA attenuated the MIWI-TDRKH interaction in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cells. OE overexpression. Quantification of blot intensity of TDRKH is shown in parentheses [the one with RNase A-untreated and piRNA-free condition in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cell lysates (lane 2) is set as 1.0 after normalization with MIWI blotting]. g Schematic diagram showing that piRNA loading facilitates the dissociation of MIWI from TDRKH. The results shown are representative of three independent experiments. Quantification of western blot analysis are represented as mean ± SD. Source data are provided as a Source Data file.

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