D incubated for 1 hour at 4uC. The beads were precipitated and washed for 10 minutes with the RIPA modified lysis buffer. Washing was repeated four times. All steps were performed with mild Title Loaded From File agitation. SDS sample buffer was added to the beads after the last wash, and then the samples were boiled, separated by SDS-PAGE and either immunoblotted with the appropriate antibodies or stained with the Coomassie blue based sensitive staining (Imperial protein stain, Pierce) according to the manufacturer’s instructions. For mass spectrometric analysis, the protein bands were excised from the stained gel and delivered to the Biological Mass Spectrometry Facility at Weizmann Institute of Science or to the University of Kentucky proteomics core facility. For Western blot analysis, protein samples were separated on 12 SDS-polyacrylamide gels, then transferred to Title Loaded From File nitrocellulose membranes (BioTrace NT, Pall Inc.). The efficiency of transfer was monitored by Ponceau-S (Sigma) staining. The membranes were blocked for 1 h at RT with 5 milk (Sigma) in TTBS. Incubation with the primary antibodies was for 1 h at RT or overnight at 4uC. The membranes were washed three times with TTBS for 5 minutes each then incubated with secondary antibody for 1 hour at room temperature and subsequently washed with TTBS four times for 5 minutes. Blots were exposed and developed using the ECL blot detection reagent EZ-ECL (Biochemical Industries) using Chemi Doc XRS+ digital camera with Image Lab software. Western blot analyses for CaM in cell lysates were performed similarly except that PVDF membranes were used (with 20 mg of protein) and the membrane was developed using NBT/BCIP (Sigma).RACE-PCR-SMARTRACE cDNA amplification kit (Clontech) was used to amplify potential CaM KMT transcripts from lymphoblastoid cells of patients and normal controls. For the first strand synthesis we used the universal primer mix (UPM) as the forward primer and the reverse primer was designed at the border of the 5th and 6th exons of the long variant, to avoid priming in the residual DNA that may be retained in the RNA preparation: 59GCACATTTCTGATGGCCTTTTCATTCC39.The product was then amplified using nested PCR reaction with UPM and a reverse primer that was located within the 4th exon of the long variant (presented in the RT-PCR paragraph). The RACE products were cloned into pGEM-T easy vector (Promega) and transformed into DH5a strain of E.coli.Fluorescence ImagingCells were grown on cover slips, fixed with freshly prepared 4 paraformaldehyde/PBS for 15 minutes, then washed extensively in PBS and mounted with Prolong Gold antifade reagent containing DAPI (Invitrogene) on microscope slides. The samples were visualized on a Leica DMR compound microscope equipped for immunofluorescence and photographed with a Spot RT digital camera (Diagnostic Instruments). Confocal fluorescent images were obtained by a Zeiss LSM Axiovert 100 laser scanning microscope.Whole Cell Protein ExtractionCells were collected by scraping, pelleted by centrifugation and washed with cold PBS three times before lysis. To stabilize transient and weak protein-protein interaction, the cells used for immunoprecipitation were treated with formaldehyde (Sigma) 1 for 15 minutes and quenched with 1.25 M glycine/PBS prior to collection [11]. The cells (16108 cells) were lysed in 1 ml modified RIPA buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 12926553 1 NP40, 1 mM EDTA, protease inhibitors (Sigma)) for 20 min on ice, followed by centrifugat.D incubated for 1 hour at 4uC. The beads were precipitated and washed for 10 minutes with the RIPA modified lysis buffer. Washing was repeated four times. All steps were performed with mild agitation. SDS sample buffer was added to the beads after the last wash, and then the samples were boiled, separated by SDS-PAGE and either immunoblotted with the appropriate antibodies or stained with the Coomassie blue based sensitive staining (Imperial protein stain, Pierce) according to the manufacturer’s instructions. For mass spectrometric analysis, the protein bands were excised from the stained gel and delivered to the Biological Mass Spectrometry Facility at Weizmann Institute of Science or to the University of Kentucky proteomics core facility. For Western blot analysis, protein samples were separated on 12 SDS-polyacrylamide gels, then transferred to nitrocellulose membranes (BioTrace NT, Pall Inc.). The efficiency of transfer was monitored by Ponceau-S (Sigma) staining. The membranes were blocked for 1 h at RT with 5 milk (Sigma) in TTBS. Incubation with the primary antibodies was for 1 h at RT or overnight at 4uC. The membranes were washed three times with TTBS for 5 minutes each then incubated with secondary antibody for 1 hour at room temperature and subsequently washed with TTBS four times for 5 minutes. Blots were exposed and developed using the ECL blot detection reagent EZ-ECL (Biochemical Industries) using Chemi Doc XRS+ digital camera with Image Lab software. Western blot analyses for CaM in cell lysates were performed similarly except that PVDF membranes were used (with 20 mg of protein) and the membrane was developed using NBT/BCIP (Sigma).RACE-PCR-SMARTRACE cDNA amplification kit (Clontech) was used to amplify potential CaM KMT transcripts from lymphoblastoid cells of patients and normal controls. For the first strand synthesis we used the universal primer mix (UPM) as the forward primer and the reverse primer was designed at the border of the 5th and 6th exons of the long variant, to avoid priming in the residual DNA that may be retained in the RNA preparation: 59GCACATTTCTGATGGCCTTTTCATTCC39.The product was then amplified using nested PCR reaction with UPM and a reverse primer that was located within the 4th exon of the long variant (presented in the RT-PCR paragraph). The RACE products were cloned into pGEM-T easy vector (Promega) and transformed into DH5a strain of E.coli.Fluorescence ImagingCells were grown on cover slips, fixed with freshly prepared 4 paraformaldehyde/PBS for 15 minutes, then washed extensively in PBS and mounted with Prolong Gold antifade reagent containing DAPI (Invitrogene) on microscope slides. The samples were visualized on a Leica DMR compound microscope equipped for immunofluorescence and photographed with a Spot RT digital camera (Diagnostic Instruments). Confocal fluorescent images were obtained by a Zeiss LSM Axiovert 100 laser scanning microscope.Whole Cell Protein ExtractionCells were collected by scraping, pelleted by centrifugation and washed with cold PBS three times before lysis. To stabilize transient and weak protein-protein interaction, the cells used for immunoprecipitation were treated with formaldehyde (Sigma) 1 for 15 minutes and quenched with 1.25 M glycine/PBS prior to collection [11]. The cells (16108 cells) were lysed in 1 ml modified RIPA buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 12926553 1 NP40, 1 mM EDTA, protease inhibitors (Sigma)) for 20 min on ice, followed by centrifugat.
