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. 2018 Jun 4;10(6):180. doi: 10.3390/cancers10060180

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© 2018 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Over-expressing recombinant KIAA0100 protein in HEK293 cells. KIAA0100 over-expressing cell line HEK293/pKIA was established by transfecting KIAA0100 recombinant plasmid to HEK293 cells: (A) cell morphology comparison between HEK293 and HEK293/pKIA. 24 and 48 h after seeding same number of cells to the culture plate; (B) Western blot confirmed over-expression of the KIAA0100 recombinant protein in HEK293/pKIA cells compared to the parent HEK293 cells; (C) immunofluorescence detection of the recombinant KIAA0100 and microtubule network (TUBA) in HEK293/pKIA cells. Cells with white arrow were not expressing recombinant KIAA0100, it served as control for the recombinant KIAA0100 staining; (D) recombinant KIAA0100 was captured by anti-FLAG antibody from cell lysate. The captured proteins were separated on 4–12% SDS-PAGE and stained with coomassie blue. Six gel slices were excised and labeled S1 to S6 for mass spectrometry analysis; (E) top protein calls from mass spectrometry analysis that match the molecular weight according to the gel slice positions; (F) Western blot to confirm the protein identified in the mass spectrometry analysis, IP with anti-FLAG tag antibody and anti-HSPA1A antibody compared to the corresponding controls with anti-DIG antibody as well as empty beads are presented; (G) immunofluorescence of recombinant KIAA0100 in HEK293/pKIA cells in a higher magnification, arrow point to the thread-like structure that both appears in the KIAA0100 staining and the TUBA staining; (H) immunofluorescence detection recombinant KIAA0100 and F-actin filaments detected by Phalloidin in HEK293/pKIA cells; (I) immunofluorescence of HEK293/pKIA cells stained with KIAA0100 and HSPA1A (arrow point to the potential co-localization spot). In all immunofluorescence images above, KIAA0100 showed in green; other co-stained targets showed in red color.

Over-expressing recombinant KIAA0100 protein in HEK293 cells. KIAA0100 over-expressing cell line HEK293/pKIA was established by transfecting KIAA0100 recombinant plasmid to HEK293 cells: (A) cell morphology comparison between HEK293 and HEK293/pKIA. 24 and 48 h after seeding same number of cells to the culture plate; (B) Western blot confirmed over-expression of the KIAA0100 recombinant protein in HEK293/pKIA cells compared to the parent HEK293 cells; (C) immunofluorescence detection of the recombinant KIAA0100 and microtubule network (TUBA) in HEK293/pKIA cells. Cells with white arrow were not expressing recombinant KIAA0100, it served as control for the recombinant KIAA0100 staining; (D) recombinant KIAA0100 was captured by anti-FLAG antibody from cell lysate. The captured proteins were separated on 4–12% SDS-PAGE and stained with coomassie blue. Six gel slices were excised and labeled S1 to S6 for mass spectrometry analysis; (E) top protein calls from mass spectrometry analysis that match the molecular weight according to the gel slice positions; (F) Western blot to confirm the protein identified in the mass spectrometry analysis, IP with anti-FLAG tag antibody and anti-HSPA1A antibody compared to the corresponding controls with anti-DIG antibody as well as empty beads are presented; (G) immunofluorescence of recombinant KIAA0100 in HEK293/pKIA cells in a higher magnification, arrow point to the thread-like structure that both appears in the KIAA0100 staining and the TUBA staining; (H) immunofluorescence detection recombinant KIAA0100 and F-actin filaments detected by Phalloidin in HEK293/pKIA cells; (I) immunofluorescence of HEK293/pKIA cells stained with KIAA0100 and HSPA1A (arrow point to the potential co-localization spot). In all immunofluorescence images above, KIAA0100 showed in green; other co-stained targets showed in red color.