![Figure 1. Overexpression or knockdown of hGAAP affects cell adhesion, cell detachment, and cell spreading. (A and B) Immunoblot (IB) of U2-OS cells with anti-HA Ab (A) or HeLa cells with anti-V5 Ab (B) shows expression of tagged hGAAP and hGAAP Ctmut in stable cell lines but not in cells expressing control plasmids (neo and puro). (C) Levels of endogenous hGAAP mRNA were determined by RT-PCR in U2-OS cells transfected with siRNAs for hGAAP (siRNA 1 and 2) or control siRNA. An analysis without reverse transcription (−RT) was included to control for DNA contamination. (D) U2-OS cells overexpressing hGAAP-HA were transfected with the indicated siRNAs, and hGAAP-HA expression was measured using IB with an anti-HA Ab. (E) Summary results (means ± SEM, from three independent experiments) show relative expression of hGAAP protein quantified from IB as in D. (F and G) Adherence of U2-OS (F) or HeLa cells (G) expressing the indicated hGAAPs was determined after washing cells with PBS at intervals after seeding. Results are means ± SEM, from three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test, relative to cells overexpressing hGAAP). (H–J) Adherent cells were quantified 20 min after addition of the indicated concentrations of EDTA to U2-OS (H), HeLa cells (I) overexpressing hGAAPs, or U2-OS cells treated with siRNAs (J). Results (H–J) are representative of three experiments and show means ± SEM. (K and L) U2-OS cells overexpressing hGAAPs (K) or after treatment with siRNAs (L) were seeded onto fibronectin-coated slides. Cell areas were measured after fixing cells at intervals after seeding. Results show means ± SEM for ≥35 cells and are typical of three experiments. (H–L) *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test, compared with control). (M) Traces show [Ca2+]i after addition of the Ca2+ ionophore, ionomycin (1 µM), in Ca2+-free HBS to populations of fura-2–loaded U2-OS cells expressing the indicated hGAAPs. (N) Summary results (means ± SEM from three independent experiments) show peak increases in [Ca2+]i expressed as fractions of those in matched neo cells. **, P < 0.01 by one-sample Student’s t test.](https://cdn.rupress.org/rup/content_public/journal/jcb/202/4/10.1083_jcb.201301016/5/jcb_201301016_fig1.jpeg?Expires=1771572537&Signature=qT9j7foxzPWhNp3~dJUWCA9JSam~Btxvr1-p-QFp0bBe2OrFTXUr9Q55M9TP9Yu-JwLd2ngH6MSs~igzMuFqyzL3hZm3AfUjvPl7NzpOFib~y5UAuWu1Cx-osBbNBVyKLV5tyVG6tGf9981VI1AbHKBZ9fyeuVM2RqlnpI67g7mBQYU5vZX~eHrzjsl2qxd1mRgSCXMx5fj4xwgiKEFdkaoi-t~B8mBQEtVQsKWC-QWMj9iN4BB6-elGQbJg-gG2gommH5gNEfh2uuetm8luBqx-xHhDPebX~TiN6pmhM4AYUTNk7p2QPI4lWlUaAcQ32wZ4KsLywJ-Fp3CpG1UOxw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Overexpression or knockdown of hGAAP affects cell adhesion, cell detachment, and cell spreading. (A and B) Immunoblot (IB) of U2-OS cells with anti-HA Ab (A) or HeLa cells with anti-V5 Ab (B) shows expression of tagged hGAAP and hGAAP Ctmut in stable cell lines but not in cells expressing control plasmids (neo and puro). (C) Levels of endogenous hGAAP mRNA were determined by RT-PCR in U2-OS cells transfected with siRNAs for hGAAP (siRNA 1 and 2) or control siRNA. An analysis without reverse transcription (−RT) was included to control for DNA contamination. (D) U2-OS cells overexpressing hGAAP-HA were transfected with the indicated siRNAs, and hGAAP-HA expression was measured using IB with an anti-HA Ab. (E) Summary results (means ± SEM, from three independent experiments) show relative expression of hGAAP protein quantified from IB as in D. (F and G) Adherence of U2-OS (F) or HeLa cells (G) expressing the indicated hGAAPs was determined after washing cells with PBS at intervals after seeding. Results are means ± SEM, from three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test, relative to cells overexpressing hGAAP). (H–J) Adherent cells were quantified 20 min after addition of the indicated concentrations of EDTA to U2-OS (H), HeLa cells (I) overexpressing hGAAPs, or U2-OS cells treated with siRNAs (J). Results (H–J) are representative of three experiments and show means ± SEM. (K and L) U2-OS cells overexpressing hGAAPs (K) or after treatment with siRNAs (L) were seeded onto fibronectin-coated slides. Cell areas were measured after fixing cells at intervals after seeding. Results show means ± SEM for ≥35 cells and are typical of three experiments. (H–L) *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test, compared with control). (M) Traces show [Ca2+]i after addition of the Ca2+ ionophore, ionomycin (1 µM), in Ca2+-free HBS to populations of fura-2–loaded U2-OS cells expressing the indicated hGAAPs. (N) Summary results (means ± SEM from three independent experiments) show peak increases in [Ca2+]i expressed as fractions of those in matched neo cells. **, P < 0.01 by one-sample Student’s t test.
