Figure 4. Reversible phosphorylation of... | Rockefeller University Press

Figure 4.

$Figure 4. Reversible phosphorylation of TRAMM is important for its mitotic function. (A) HeLa cells were untreated (Async, asynchronous) or treated with colcemid (Colc). Lysates were fractionated on a Superose 6 size exclusion column and probed for TRAMM. The migration of molecular size standards is indicated above the top blot. The location of the TRAPP complex was determined by Western analysis of TrappC11 (not depicted) and by a previous study using the same column (Bassik et al., 2013). Black lines indicate that intervening lanes have been spliced out. TL, total lysate. (B) Lysates were prepared from HeLa cells that were either untreated (asynchronous), treated with thymidine (Thym), or treated with colcemid. A portion of each lysate was left untreated (−) or treated with λ-phosphatase (λ-PPase; +) before Western analysis. (C) Treatment of cells in B was repeated using HeLa, A549, and HT1080 cells. p-Hist-H3, phospho–histone H3. (D) HeLa cells were arrested at the G1/S boundary by treatment with thymidine, and then, the cells were transferred to regular growth medium containing nocodazole. Samples were removed at the times indicated (hours) and examined by Western analysis. (E) HeLa cells were untreated or treated with either colcemid, RO-3306, or both colcemid and RO-3306, as indicated. Cells were then lysed, and proteins were detected by Western analysis. (F) HeLa cells were arrested in prometaphase by treatment with nocodazole and released into regular growth medium. Samples were removed at the times indicated (hours) and examined by Western analysis. (G) HeLa cells were treated with NS or TRAMM-specific siRNA. In some cases, the cells were cotransfected with a plasmid expressing an siRNA-resistant form of either wild-type (WT) TRAMM, TRAMM-5A, or TRAMM-5D, as indicated. Results are shown ± SD, and significance was assessed by an unpaired t test. KD, knockdown.$

Reversible phosphorylation of TRAMM is important for its mitotic function. (A) HeLa cells were untreated (Async, asynchronous) or treated with colcemid (Colc). Lysates were fractionated on a Superose 6 size exclusion column and probed for TRAMM. The migration of molecular size standards is indicated above the top blot. The location of the TRAPP complex was determined by Western analysis of TrappC11 (not depicted) and by a previous study using the same column (Bassik et al., 2013). Black lines indicate that intervening lanes have been spliced out. TL, total lysate. (B) Lysates were prepared from HeLa cells that were either untreated (asynchronous), treated with thymidine (Thym), or treated with colcemid. A portion of each lysate was left untreated (−) or treated with λ-phosphatase (λ-PPase; +) before Western analysis. (C) Treatment of cells in B was repeated using HeLa, A549, and HT1080 cells. p-Hist-H3, phospho–histone H3. (D) HeLa cells were arrested at the G1/S boundary by treatment with thymidine, and then, the cells were transferred to regular growth medium containing nocodazole. Samples were removed at the times indicated (hours) and examined by Western analysis. (E) HeLa cells were untreated or treated with either colcemid, RO-3306, or both colcemid and RO-3306, as indicated. Cells were then lysed, and proteins were detected by Western analysis. (F) HeLa cells were arrested in prometaphase by treatment with nocodazole and released into regular growth medium. Samples were removed at the times indicated (hours) and examined by Western analysis. (G) HeLa cells were treated with NS or TRAMM-specific siRNA. In some cases, the cells were cotransfected with a plasmid expressing an siRNA-resistant form of either wild-type (WT) TRAMM, TRAMM-5A, or TRAMM-5D, as indicated. Results are shown ± SD, and significance was assessed by an unpaired t test. KD, knockdown.