Fig 1: PON2 and RAB4A promoted LRIG1-mediated down-regulation of PDGFRA expression. In the triple co-transfection system, cells were co-transfected with LRIG1, PDGFRA, and individual shRNA vectors targeting the LRIG1 interactors RAB4A and PON2 as described in the legend to Fig. 1. A, representative Western blots showing RAB4A, PON2, and PDGFRA expression in HEK293 cells triple–co-transfected with LRIG1, PDGFRA, and different shRNAs targeting RAB4A (left) or PON2 (right). Vertical black lines, non-adjacent lanes on the same blot. B, quantification of RAB4A (left) and PON2 (right) protein levels after triple co-transfections with LRIG1, PDGFRA, and the indicated shRNAs. The results from four independent experiments similar to A are shown. C, quantification of PDGFRA protein levels from four biological replicates as described in B. Darker and lighter gray bars represent the upper and lower PDGFRA bands, respectively. Statistical analyses were performed as described in the legend to Fig. 1. Error bars, S.D.
Fig 2: Effects of PON2 and ZBTB16 down-regulation on LRIG1-deficient cells. LRIG1-null HEK293T cells were transiently triple-transfected with the pcDNA3.1 control vector (A) or LRIG1 (B) together with PDGFRA and one of the following shRNA mixtures: non-targeting shRNAs (CTRL), shRNAs targeting PON2, or shRNAs targeting ZBTB16. The relative PDGFRA levels observed 4 days after transfection by Western blotting are shown; actin served as the reference protein. The experiment was repeated four independent times. Error bars, S.D. Student's t test was used for statistical analyses (*, p < 0.05; **, p < 0.01).
Fig 3: Subcellular localization of PON2 and LRIG1 in transfected HEK293 cells. HEK293 cells were triple–co-transfected with LRIG1, PDGFRA, and shRNA targeting PON2 or control shRNA; stained with antibodies against PON2 and LRIG1; and then analyzed using laser confocal microscopy. A, PON2 staining of cells transfected with the non-target control shRNA (left), shRNA D7 targeting PON2 (middle), or shRNA D8 targeting PON2 (right). B, quantifications of four experiments similar to A. Error bars, S.D. **, p < 0.01. C, HEK293 cells were triple–co-transfected with LRIG1, PDGFRA, and non-target control shRNA and stained with DAPI and antibodies against Myc (LRIG1) and PON2. Top left, DAPI staining of cell nuclei (blue). Bottom left, PON2 staining (red). Top middle, LRIG1 (Myc) staining (green). Bottom middle, LRIG1 and PON2 co-localization (white). Right, overlay of DAPI, LRIG1, and PON2 staining. Scale bar, 10 μm.
Fig 4: Young NHP brain has no sex bias in PON2 isoforms expression in STR and SN regions (n = 5). Protein expression of PON2 isoforms, i.e., 39 kDa and 41 kDa in STR (A, B) and SN (C, D), respectively. Representative blot showing PON2 isoforms expression in STR (E), and SN (F). Corresponding image of total protein in STR (G) and SN (H). Optical density of UCP5 and UCP4 bands were normalized to total protein per lane. The normality of infant male and female data was confirmed by a Shapiro–Wilk test. 39 kDa PON2 STR Male (W = 0.97, p = 0.89) Female (W = 0.91, p = 0.50); 41 kDa PON2 Male (W = 0.96, p = 0.87) Female (W = 0.80, p = 0.09); 39 kDa PON2 Male (W = 0.84, p = 0.18) Female (W = 0.83, p = 0.15); 41 kDa PON2 SN Male (W = 0.97, p = 0.87) Female (W = 0.97, p = 0.90). Data were expressed as mean ± SEM. Male and female data in each region was compared by two-tailed unpaired Student's t test. 39 kDa PON2 STR (two-tailed t (8) = 0.07, p = 0.94), 41 kDa PON2 STR (two-tailed t (8) = 0.0, p > 0.99), 39 kDa PON2 SN (two-tailed t (8) = 0.03, p = 0.97), 41 kDa PON2 SN (two-tailed t (8) = 0.0, p > 0.99)
Fig 5: Adult NHP brain has sex bias in PON2 isoforms expression in STR and SN regions. STR (n = 5) and SN (n = 4). Protein expression of PON2 isoforms, i.e., 39 kDa and 41 kDa in STR (A, B) and SN (C, D), respectively. Representative blot showing PON2 isoforms expression in STR (E), and SN (F). Corresponding image of total protein in STR (G) and SN (H). Optical density of UCP5 and UCP4 bands were normalized to total protein per lane. The normality of infant male and female data was confirmed by a Shapiro–Wilk test. 39 kDa PON2 STR Male (W = 0.84, p = 0.17) Female (W = 0.97, p = 0.87); 41 kDa PON2 Male (W = 0.95, p = 0.77) Female (W = 0.92, p = 0.54); 39 kDa PON2 Male (W = 0.92, p = 0.58) Female (W = 0.91, p = 0.51); 41 kDa PON2 SN Male (W = 0.87, p = 0.33) Female (W = 0.95, p = 0.73). Data were expressed as mean ± SEM. Male and female data in each region was compared by two-tailed unpaired Student's t test. 39 kDa PON2 STR (two-tailed t (6) = 3.75, p = 0.0095), 41 kDa PON2 STR (two-tailed t (8) = 4.87, p = 0.0012), 39 kDa PON2 SN (two-tailed t (8) = 0.03, p = 0.97), 41 kDa PON2 SN (two-tailed t (6) = 2.72, p = 0.034). Asterisks indicate statistical significance in comparison with vehicle group, *p < 0.05, **p < 0.01, ***p < 0.001
Supplier Page from Abcam for Anti-PON2 antibody [EPR15295-82]