![Figure 6. Increased PTEN cleavage, reduced phosphorylation, stability, and activity of PTEN in ROCK1−/− BMMs. (A) Equal amounts of lysates from WT and ROCK1−/− BMMs were subjected to Western blot analysis using an anti-PTEN antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated ROCK1−/− BMMs, respectively. Top arrow represents total PTEN protein, whereas the middle arrow and stars indicate cleaved PTEN products in WT and ROCK1−/− BMMs (n = 3). The same blot was reprobed for β-actin to show protein loading in each lane (bottom panel). (B) Equal amounts of lysates were subjected to Western blot analysis using an anti-phospho PTEN antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated ROCK1−/− BMMs, respectively. The top arrow (on left) indicates the level of phospho PTEN levels in WT and ROCK1−/− BMMs; bottom arrow indicates total β-actin levels in each lane. Western blot shown is representative of at least 3 independent experiments. The bar graph indicates the band intensity of phospho PTEN (n = 4). (C) PTEN activity assay. WT and ROCK1−/− BMMs cultured for 10 days in the presence of M-CSF were starved overnight and stimulated with 100 ng/mL M-CSF for 5 minutes. Equal amount of lysates were subjected to PTEN activity assay using the malachite green assay kit. PTEN activity is expressed as the amount of phosphate released. Data shown are the mean value of phosphate release in 3 independent experiments (n = 3, *P < .05). (D) Pulse-chase analysis of PTEN stability. WT and ROCK1−/− BMMs were metabolically labeled with [35S]-methionine. Medium was replaced with methionine-containing chase medium at time zero and chased at the indicated time points. PTEN was immunoprecipitated from labeled cell extracts, separated by gel electrophoresis, and detected by autoradiography. Lanes 1 to 3 represent PTEN expression in WT BMMs chased at different time points; lanes 4 to 6 indicate expression of PTEN in ROCK1−/− BMMs chased for the same amount of time as WT cells (n = 2).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/9/10.1182_blood-2009-08-237222/4/zh89990948520006.jpeg?Expires=1735808484&Signature=q8mlXvuyPCrPWjTkdwMsskcX7nQ0gTP0Sqecm1BSKo1VyeNKVR2jM-BKmRFCGy-dgzXCj8GB9qloMSHYGsa3mB4N6bBssNsCfGTblGj0ns6qp~8i295arpOxDRKMLCsEGzjbBSk9MsPNvXHxmgLUegfP~adCcxQza47dtaqyyzZRK~LBe1qXcEa3y5T1j3f~MHzcfnxNZHaTPteI0Lf2zYJY~M~JsRIxwqeHUXg9nENEfUJVRkY5I1c~hoAylxIznMz9EguJMpk289V4pj--5F99DJNfYENDyOOfsTSsRJskGPn~dpSbXjjxPQHK03q0QbYeb9zWyFLnHfYXfVDGGg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Increased PTEN cleavage, reduced phosphorylation, stability, and activity of PTEN in ROCK1−/− BMMs. (A) Equal amounts of lysates from WT and ROCK1−/− BMMs were subjected to Western blot analysis using an anti-PTEN antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated ROCK1−/− BMMs, respectively. Top arrow represents total PTEN protein, whereas the middle arrow and stars indicate cleaved PTEN products in WT and ROCK1−/− BMMs (n = 3). The same blot was reprobed for β-actin to show protein loading in each lane (bottom panel). (B) Equal amounts of lysates were subjected to Western blot analysis using an anti-phospho PTEN antibody. Lanes 1 and 2 consist of lysates derived from unstimulated and stimulated WT BMMs, respectively; lanes 3 and 4 consist of lysates derived from unstimulated and stimulated ROCK1−/− BMMs, respectively. The top arrow (on left) indicates the level of phospho PTEN levels in WT and ROCK1−/− BMMs; bottom arrow indicates total β-actin levels in each lane. Western blot shown is representative of at least 3 independent experiments. The bar graph indicates the band intensity of phospho PTEN (n = 4). (C) PTEN activity assay. WT and ROCK1−/− BMMs cultured for 10 days in the presence of M-CSF were starved overnight and stimulated with 100 ng/mL M-CSF for 5 minutes. Equal amount of lysates were subjected to PTEN activity assay using the malachite green assay kit. PTEN activity is expressed as the amount of phosphate released. Data shown are the mean value of phosphate release in 3 independent experiments (n = 3, *P < .05). (D) Pulse-chase analysis of PTEN stability. WT and ROCK1−/− BMMs were metabolically labeled with [35S]-methionine. Medium was replaced with methionine-containing chase medium at time zero and chased at the indicated time points. PTEN was immunoprecipitated from labeled cell extracts, separated by gel electrophoresis, and detected by autoradiography. Lanes 1 to 3 represent PTEN expression in WT BMMs chased at different time points; lanes 4 to 6 indicate expression of PTEN in ROCK1−/− BMMs chased for the same amount of time as WT cells (n = 2).
