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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Cell Signal. 2015 Oct 19;28(1):43–52. doi: 10.1016/j.cellsig.2015.10.004

Figure 2. LARG is phosphorylated and dephosphorylated during mitosis.

Figure 2

(A) LARG is dephosphorylated as cells exit mitosis. HeLa cells arrested in prometaphase by nocodazole were collected by mitotic shake-off and re-plated on poly-L-lysine coated plates in fresh media without nocodazole to release the cells from mitotic arrest. Cells were lysed at the indicated times after release, and lysates were immunoblotted using anti-LARG, anti-cyclin B1 and anti-HSP90 (loading control) antibodies. (B) LARG is rapidly phosphorylated after release from Cdk1 inhibitor RO-3306. Cells were synchronized with RO-3306, released from G2/M block by washing and incubating in media without RO-3306, and lysed at indicated times. Cell lysates were immunoblotted using anti-LARG and anti-HSP90 (loading control) antibodies. (C) LARG is phosphorylated coordinate with Cyclin B1 accumulation and is dephosphorylated in late mitosis. HeLa cells were treated with 2 mM thymidine for 16 hrs to block the cells in S phase, followed by release in normal growth medium for 8 hrs and subsequent thymidine treatment for 16 hrs. HeLa cells were then released from S-phase block into normal growth media and collected at indicated time points. Cell lysates were immunoblotted using anti-LARG, anti-cyclin B1, anti-GAPDH (loading control) antibodies.