Journal: The Journal of Cell Biology

Article Title: Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

doi: 10.1083/jcb.201306083

Figure Lengend Snippet: ADD1 phosphorylation at S12 and S355 is required for its interaction with the motor domain of Myo10. (A) The cell lysates prepared from HeLa cells or those stably expressing FLAG-ADD1 were incubated with anti-FLAG M2 affinity resins. The bound proteins were eluted from the resins with FLAG peptides and analyzed by immunoblotting (IB) with anti-Myo10 or anti-FLAG. WCL, whole-cell lysates. (B) GFP-fused Myo10 (GFP-Myo10) and mutants were transiently expressed in HEK293 cells and were analyzed by immunoblotting with anti-GFP. The scheme shows the domain structures of GFP-Myo10. CC, coiled-coil domain; PEST, polypeptide enriched in proline, glutamic acid, serine, and threonine residues. (C and D) FLAG-ADD1 was transiently coexpressed with GFP-Myo10 or its mutants in HEK293 cells. GFP-Myo10 was immunoprecipitated (IP) by anti-GFP, and the immunocomplexes were analyzed by immunoblotting with anti-FLAG or anti-GFP. (E) GFP-Myo10-N was transiently coexpressed with (+) or without (−) the FLAG-ADD1Δtail mutant in HEK293 cells. The cell lysates were incubated with anti-FLAG, and the immunocomplexes were analyzed by immunoblotting with anti-GFP or anti-FLAG. (F) GFP-Myo10-N was transiently coexpressed with FLAG-ADD1 or its mutants in HEK293 cells. FLAG-ADD1 was immunoprecipitated by anti-FLAG, and the immunocomplexes were analyzed by immunoblotting with anti-GFP or anti-FLAG. (G) HeLa cells were infected with lentiviruses expressing shRNAs to Myo10 (sh-Myo10 #1 and sh-Myo10 #2) or to luciferase (sh-Luc.) as a control. The expression levels of Myo10, ADD1, and β-tubulin (as a loading control) were analyzed by immunoblotting with the indicated antibodies. (H) The cells, as in G, were stained for ADD1, α-tubulin, and DNA. Arrows indicate misaligned chromosomes. The percentage of ADD1 association with mitotic spindles in the total number of mitotic cells counted was measured ( n > 270). Values (means ± SD) are from three independent experiments. **, P < 0.01. Bars, 5 µm.

Article Snippet: The mouse anti-FLAG (M2), rabbit anti-FLAG, mouse anti–α-tubulin (DM1A), mouse anti-GFP (B-2), mouse anti–β-actin antibodies, and nocodazole were purchased from Sigma-Aldrich.

Techniques: Stable Transfection, Expressing, Incubation, Western Blot, Immunoprecipitation, Mutagenesis, Infection, Luciferase, Staining

Journal: Scientific Reports

Article Title: The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape

doi: 10.1038/s41598-023-28177-z

Figure Lengend Snippet: AK1 and AK2 in testis, spermatozoa, flagellar proteins ( A ) and immunoprecipitation (IP) ( B ) . + / + : Odf4 +/+ . −/−: Odf4 −/− . Western blotting. ( A , B ) Cropped images. ( A ) (Left) Testicular and cauda epididymal sperm extracted samples. β-ACTIN and β-TUBULIN: internal controls. AK1 and AK2 are detected in both Odf4 +/+ testes (Top) and cauda spermatozoa (Bottom); however, they are detected in Odf4 +/+ , but reduced or hardly detected in Odf4 −/− cauda spermatozoa (Bottom). Further cropped images are shown in the supplementary Fig S6 for testis (Top) and spermatozoon (Bottom). These original full-length images with different exposure times are shown in the supplementary SD2 for testis (Top) and SD3 spermatozoon (Bottom). (Right) All flagellar proteins extracted from Odf4 +/+ and Odf4 −/− mice examined are immunopositive for each corresponding antibody: ODF1, ODF2, TEKTIN4, CATSPER3, AQP3, AQP7, AQP8, SEPTIN4, SEPTIN7, SLC22A14, GAPDS, GAPDH, β-ACTIN and β-TUBULIN. Further cropped images are shown in the supplementary Fig S7. These original full-length images with different exposure times are shown in each corresponding supplementary SD from SD4 to SD13. ( B ) Cropped images of IP with anti-GFP antibody for the extracts in RIPA buffer from Tg(Odf4-Egfp) and Odf4 −/− (control) spermatozoa and western blotting with anti AK1 (23 kDa) and AK2 (30 kDa) antibodies for the same blotted membrane after SDS-PAGE. Lanes 1 and 4 (Tg): Tg(Odf4-Egfp) sperm extracts. Lanes 2 and 5: Odf4 −/− (−/−) sperm extracts. Lanes 3 and 6: Positive control (IP-untreated Odf4 +/+ samples) to identify AK1 and AK2, respectively. The AK1 and AK2 signals are clearly detected in lanes 1 and 4 for Tg and lanes 3 and 6 for the positive control, but they are not found in lanes 2 and 5 for Odf4 −/− (−/−), respectively (rectangles). These original full-length images with different exposure times are shown in the supplementary SD14. H on the right side indicates the position of the IgG heavy chain (approximately 55 kDa). M, marker proteins (protein molecular marker and/or prestained marker).

Article Snippet: The same amount of samples for each pair was subjected to IP, which was performed using a DynabeadsTM Protein G Immunoprecipitation Kit with a mouse monoclonal antibody against GFP (B2: IgG 2a , Santa Cruz Biotechnology Inc.).

Techniques: Immunoprecipitation, Western Blot, Control, Membrane, SDS Page, Positive Control, Marker