Fig 1: A: Quantification of CDKN1B mRNA expression by qPCR. Total RNA was extracted from peripheral blood. White blood cells from peripheral blood were spun down, lysed, and total RNA was purified using the Qiagen RNA easy extraction kit (cat. 52304). RNA was reversed transcribed, and quantitative PCR was carried using the Qiagen one-step SYBR green RT-PCR kit. Q-PCR primers (primer sequences are detailed in Supp. Fig. S4B) spanned the 3′ end of exon 1 (forward) of CDKN1B to the 5′ end of exon 2 (reverse). The amount of CDKN1B detected was normalized against the ubiquitously expressed ABL1 gene and expressed as a relative value Ratio (ABL1/CDKN1B) = 2CT(ABL1)-CT(CDKN1B). B: Quantification of CDKN1B minimal promoter activity. Region incorporating the 5′UTR of CDKN1B (encompassing the −79 and −73 residues) were amplified from patient DNA and subcloned into the pGL3 basic luciferase reporter vector (Promega cat. E1751). One microgram of pGL3−79C-73G, pGL3−79T-79G, pGL3−79C-73A, or an empty pGL3 vector was cotransfected with 20 ng of pRL-TK (Renilla reporter construct, Promega cat. E2241) into HEK293T cells with the ProFection mammalian transfection system (Promega cat. E1200) according to the manufacturer's protocol. Cells were transfected for 48 hr, followed by a 12 hr period of serum starvation. The dual-luciferase reporter assay system (Promega cat. E1910) was used to assay relative luciferase activity. The data represent the mean of three independent experiments with standard error bars. C: Western blot analysis of CDKN1B from white blood cells. Protein samples were extracted from peripheral blood samples by first isolating white blood cells through centrifugation. These cells were subsequently lysed using RIPA lysis buffer (50 mM Tris HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) and run on a 12% SDS-PAGE. Western blotting was carried out using an anti-p27 antibody (BD Biosciences cat. 610242) and an anti-β-tubulin antibody (Cell Signaling cat. 2128) (left). Relative intensity of the signal obtained from the western blot was quantified using ImageJ (NIH). Bar chart shows the average of signals (and standard error) quantified from three blots normalized to β-tubulin level in each independent blood sample (right).