Figure 4
Figure 4. DDR1a is the major isoform expressed in Hodgkin lymphoma cell lines and is regulated by LMP1 in GC B cells. (A) PCR of cDNA from 2 representative Hodgkin lymphoma cell lines, L428 and L591 revealed the presence of a single band for 5 of 6 primer pairs spanning the coding region of DDR1. Multiple bands were detected by primer pair 4 spanning the entire juxtamembraneous domain. Sequencing of the PCR product of primer pair 4 revealed two overlapping sequences consistent with the presence of the DDR1a isoform and the DDR1d isoform. (B) To confirm these observations, a second set of primers was optimized to amplify only the juxtamembraneous domain. The amplified product was subjected to gel electrophoresis and individual bands sequenced from the gel. The strongest band present was found to correspond to the kinase-active DDR1a isoform. Transcripts for the kinase inactive form, DDR1d, as well as those encoding several potential novel isoforms were also present (supplemental Figure 3). Shown here are data from L591 cells, although similar results were obtained for L428 cells. (C) An isoform-specific Q-RT-PCR assay confirmed the expression of the DDR1a isoform in Hodgkin lymphoma cell lines. Normalized values were expressed relative to L540 set to a value of 1. (D) Q-RT-PCR analysis revealed that LMP1 increased the expression of DDR1a, but not the other kinase-active isoform, DDR1b, in human GC B cells. Normalized values were expressed relative to empty vector set to a value of 1.

DDR1a is the major isoform expressed in Hodgkin lymphoma cell lines and is regulated by LMP1 in GC B cells. (A) PCR of cDNA from 2 representative Hodgkin lymphoma cell lines, L428 and L591 revealed the presence of a single band for 5 of 6 primer pairs spanning the coding region of DDR1. Multiple bands were detected by primer pair 4 spanning the entire juxtamembraneous domain. Sequencing of the PCR product of primer pair 4 revealed two overlapping sequences consistent with the presence of the DDR1a isoform and the DDR1d isoform. (B) To confirm these observations, a second set of primers was optimized to amplify only the juxtamembraneous domain. The amplified product was subjected to gel electrophoresis and individual bands sequenced from the gel. The strongest band present was found to correspond to the kinase-active DDR1a isoform. Transcripts for the kinase inactive form, DDR1d, as well as those encoding several potential novel isoforms were also present (supplemental Figure 3). Shown here are data from L591 cells, although similar results were obtained for L428 cells. (C) An isoform-specific Q-RT-PCR assay confirmed the expression of the DDR1a isoform in Hodgkin lymphoma cell lines. Normalized values were expressed relative to L540 set to a value of 1. (D) Q-RT-PCR analysis revealed that LMP1 increased the expression of DDR1a, but not the other kinase-active isoform, DDR1b, in human GC B cells. Normalized values were expressed relative to empty vector set to a value of 1.

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