![Figure 7. The phosphorylation status of ribosomal protein P2 in AML cell lines and patient blasts. The effect of anthracycline in vitro and in vivo. (A,B) IPC-81 cells were treated for 2.5 hours with 0.5 μmol/L DNR, the last 90 minutes of which with 32Pi. Proteins were separated by 2DE (pI 4-7) and silver stained (A) or autoradiographed (B). The spots circled with a solid line were identified by MS analysis as rpP2. Note the lack of labeled phosphate in the most basic spot (right subpanel). (C) HL60 cells were pulse-labeled with [35S]methionine for 5, 10, or 60 minutes and extracts subjected to 2DE (pI 4-5) and autoradiography. The circled spots show rpP2. Note the progressive but slow shift toward the more acidic form as a function of time. (D) HL60 cells were prelabeled with [35S]methionine and chased for 6.5 hours with unlabeled methionine with vehicle (left subpanel) or 8 μmol/L DNR present during the last 6 hours of incubation. The circled spots represent rpP2 from an autoradiogram of 2DE (pI 4-5) gel. (E) Experiment as for panel D, except that gels were silver-stained and incubation time with DNR was 4 hours. (F) As for panel E, except that the phosphatase inhibitor calyculin A (CCA; 0.1 μmol/L) was present during the last 3 hours of incubation. (G) Blasts from patients with AML (M5, patient 11) were treated for 6 hours in vitro with vehicle (left subpanel) or 8 μmol/L DNR (right subpanel) and analyzed by 2DE (pI 4-5). The circled spots represent rpP2. (H) Blasts isolated from 11 patients with various AML classification (M1, M2, M4, M5, M6), one with ALL, and one with LBL were treated in vitro with DNR (8 μmol/L, 6 hours). The percentage increase of nonphosphorylated rpP2 was determined from the relative intensity of PP-rpP2, P-rpP2, and nonphospho-rpP2 in silver-stained gels like the one shown in panel G. (I) AML blasts were isolated from a patient (M4, patient 8) before (left subpanel) and 4 hours after (right subpanel) the onset of induction treatment with IDA and cytarabine and analyzed by 2DE (pI 4-5). Note the significant formation of the monophosphorylated form of rpP2 after induction treatment.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/111/5/10.1182_blood-2007-07-103242/2/zh80060816500007.jpeg?Expires=1735817163&Signature=17Z3oxJUl9z23Y3pYl-S3VP6~16KBf7N2wzC2gRIv8bKVZXePiqCd1rS8A8949C2YKBvKv0x7viIECzPPYC67BeRbqx3ar7~pVl3X2io4~PaEY9JyaXkt~rm6fLO2rL6mlpO28PuIzawS8SVC~mSsFFkZjAbjwt722Pa~wOxrPElvWymEZhG5GmPRK-pVK56V4s4UqzrajDAkucSOfYLQ24mbqC~wmtkDpGp4e5C3VEGGprWneXLFs7Q0fz-Xm9Dr8X7r56-~QAxXzkNM3rB0RD85aaGiE23ECrFzSOQNCID77w0~RZ-zyblL5POwuO5bYmiy7169PnqZqwXEA762A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The phosphorylation status of ribosomal protein P2 in AML cell lines and patient blasts. The effect of anthracycline in vitro and in vivo. (A,B) IPC-81 cells were treated for 2.5 hours with 0.5 μmol/L DNR, the last 90 minutes of which with 32Pi. Proteins were separated by 2DE (pI 4-7) and silver stained (A) or autoradiographed (B). The spots circled with a solid line were identified by MS analysis as rpP2. Note the lack of labeled phosphate in the most basic spot (right subpanel). (C) HL60 cells were pulse-labeled with [35S]methionine for 5, 10, or 60 minutes and extracts subjected to 2DE (pI 4-5) and autoradiography. The circled spots show rpP2. Note the progressive but slow shift toward the more acidic form as a function of time. (D) HL60 cells were prelabeled with [35S]methionine and chased for 6.5 hours with unlabeled methionine with vehicle (left subpanel) or 8 μmol/L DNR present during the last 6 hours of incubation. The circled spots represent rpP2 from an autoradiogram of 2DE (pI 4-5) gel. (E) Experiment as for panel D, except that gels were silver-stained and incubation time with DNR was 4 hours. (F) As for panel E, except that the phosphatase inhibitor calyculin A (CCA; 0.1 μmol/L) was present during the last 3 hours of incubation. (G) Blasts from patients with AML (M5, patient 11) were treated for 6 hours in vitro with vehicle (left subpanel) or 8 μmol/L DNR (right subpanel) and analyzed by 2DE (pI 4-5). The circled spots represent rpP2. (H) Blasts isolated from 11 patients with various AML classification (M1, M2, M4, M5, M6), one with ALL, and one with LBL were treated in vitro with DNR (8 μmol/L, 6 hours). The percentage increase of nonphosphorylated rpP2 was determined from the relative intensity of PP-rpP2, P-rpP2, and nonphospho-rpP2 in silver-stained gels like the one shown in panel G. (I) AML blasts were isolated from a patient (M4, patient 8) before (left subpanel) and 4 hours after (right subpanel) the onset of induction treatment with IDA and cytarabine and analyzed by 2DE (pI 4-5). Note the significant formation of the monophosphorylated form of rpP2 after induction treatment.
