Skip to main content
. 2021 Mar 4;11:5266. doi: 10.1038/s41598-021-84624-9

Figure 4.

Figure 4

p19 associates with CD5L to form a putative heterodimer of p19/CD5L. (a) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC and cultured for 3 days, and total cell lysates or culture supernatants were immunoprecipitated with anti-FLAG or anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-c-MYC or biotin-conjugated anti-p19, respectively. Immunoprecipitated protein was confirmed by western blotting with the antibody used for immunoprecipitation. (b) HEK293T cells were transiently transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, or both and p3 × FLAG-CMV-14-Hyper-p19/CD5L, then cultured for 3 days. The culture supernatants were then subjected to p19/CD5L-specific ELISA in triplicate. Data are shown as the mean ± SD. (c) Naive CD4+ T cells were stimulated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) under pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 8 times, and subjected to immunoprecipitation with anti-CD5L followed by western blotting with biotin-conjugated anti-p19 and subsequently anti-CD5L. (d) Naive CD4+ T cells were also similarly stimulated with plate-coated anti-CD3 and anti-CD28 under Th, Th0 and pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 10 times, followed by p19/CD5L-specific ELISA in triplicate. (e) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19WT or p3 × FLAG-CMV-14-p19C55S and pCMV3-CD5L-c-MYC and cultured for 3 days. The culture supernatants were immunoprecipitated with anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-p19, then anti-c-MYC. Data are shown as the mean ± SD and representative of four (a) or two (b–e) independent experiments. P values were determined using one-way ANOVA. *P < 0.05.