ZFIN Image: Tomasello et al., 2021, Fig. 3

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Figure Caption

Fig. 3

FAM57B interacts with CerS but does not have CerS activity

(A) CerS2 activity assayed using C24:1-CoA in CerS2 KO HEK293T cells. Statistical analysis by ttest ∗p ≤ 0.05, ∗∗p ≤ 0.01, error bars SEM. Technical experimental replicates n = 3.

(B) (Upper) Western blot analysis of total human FAM57B-Flag and CerS2-HA after transfection in HEK293T cells. Proteins were prepared from HEK293T cells overexpressing the indicated constructs. Anti-HA and anti-Flag are indicated. (Lower) CerS2 activity assayed using C24:1-CoA in HEK293T cells. GAPDH was used as a loading control. Statistical analysis by ttest ∗p ≤ 0.05, ∗∗p ≤ 0.01, error bars SEM. Technical experimental replicates n = 3.

(C) (Upper) Western blot analysis of total human FAM57B-Flag, CerS5-HA and CerS6-HA after transfection in HEK293T cells. Proteins were prepared from cells overexpressing the indicated constructs. Anti-HA and anti-Flag are indicated. (Lower) CerS5 and CerS6 activity was assayed using C16:0-CoA in HEK293T cells. Anti-HA and anti-Flag are indicated. GAPDH was used as a loading control. Technical experimental replicates n = 4.

(D) Total cell lysates were prepared from the co-transfected cells with FAM57B-Flag and CerS2, 5 or 6-HA constructs and solubilized with 1% NP-40. Total lysates (input) or proteins immuno-precipitated with anti-Flag M2 agarose (IP) were subjected to immunoblotting with anti-HA or anti-Flag antibodies. GAPDH was used as a loading control. Technical experimental replicates n = 3.

Acknowledgments

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