carf  (Cell Signaling Technology Inc)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF is highly expressed in spermatocytes and spermatids of the testis (A) NCBI database analysis of the expression profile of Carf mRNA in mouse tissues. (B) qPCR analyses of Carf mRNA levels in multiple organs of mice. Data are presented as the mean ± SEM, n = 3. (C) Immunofluorescence staining analysis of CARF (green) in testis sections. γH2AX (red) was used as a marker for spermatocytes. The nuclei were stained with DAPI (blue). Spc indicates spermatocytes, Ser indicates Sertoli cells, Ley indicates Leydig cells, and Rspd indicates round sperm. Scale bar: 50 μm. (D) Immunofluorescence staining analysis of CARF (red) and PNA (green) in testis sections. The nuclei were stained with DAPI (blue), Scale bar: 50 μm. (E,F) The expression pattern of CARF in the mouse germline atlas was analyzed via a single-cell sequencing database (http://malehealthatlas.cn/ ).

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Sequencing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF interacts with PABPC1 to participate in RNA alternative splicing (A) Abnormal alternative splicing patterns, including skipped exons, alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI) caused by Carf defects. (B) Abnormal variable splicing of the functional gene Hira, which is related to germ cell development caused by Carf defects. It belongs to the alternative 5′ splice site (A5SS) exception mode. (C) Abnormal variable splicing of the functional gene Surf1, which is related to germ cell development caused by Carf defects. It is an alternative 3′ splice site (A3SS). (D) Abnormal variable splicing of the functional gene Usf2, which is related to germ cell development caused by Carf defects. It belongs to the mutually exclusive exons (MXE). (E) Abnormal variable splicing of the functional gene Lrmp, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (F) Abnormal variable splicing of the functional gene Pkd2l1, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (G) Co-IP analysis of the interaction between CARF and PABPC1. PABPC1 expression was detected in the IP products of CARF, and IgG was used as a control. GAPDH served as a loading control. (H) Co-IP analysis of the interaction between CARF and PABPC1. CARF expression was detected in the IP products of PABPC1, and IgG was used as a control. GAPDH served as a loading control. (I) Immunostaining of PABPC1 in wild-type and Carf–/– testis sections (PABPC1: green); DAPI was used to stain the dye the nuclei, scale bar: 50 μm. (J) Western blot analysis of PABPC1 protein levels in testes from wild-type and Carf–/– mice. GAPDH served as a loading control. (K) Immunohistochemical analysis of the expression of PABPC1 in testes from wild-type and Carf–/– testis sections. Scale bar: 50 μm. (L) Quantitative results of (K). n = 3, Data are presented as the mean ± SEM. ***P < 0.001.

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Alternative Splicing, Functional Assay, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Staining, Western Blot, Immunohistochemical staining