Fig. 1

A-B Generation of lentiviral mediated stable HepG2 lines expressing Wild Type/missense variants of LDLR. A Immunofluorescence with anti-FLAG (Fg), anti-Calnexin (CNX), and anti- Na⁺K⁺ ATPase antibodies to understand the subcellular localization of FLAG-tagged (Fg) WT-LDLR, D482H-LDLR, and C667F-LDLR in stable HepG2 cells. Wild-type LDLR is expressed predominantly on the plasma membrane, whereas D482H-LDLR and C667F-LDLR are retained in the Endoplasmic Reticulum (ER). Calnexin (CNX) was used as the ER marker and Na⁺K⁺ ATPase as the marker of the plasma membrane. The color images represent merged images of red (Fg) and green (Calnexin or Na⁺K⁺ ATPase) channels. The fluorescent images were captured using the 100-X oil immersion objective of a Nikon Eclipse 2000 Confocal Microscope and color-enhanced using ImageJ software. Scale bar = 20 µm. B (i)) Western blotting with anti-FLAG antibody confirms the expression of mature (M) and precursor (P) LDLR protein of 160 kDa and 120 kDa, respectively in WT-LDLR expressing HepG2. The missense variants D482H-LDLR and C667F-LDLR fail to express the mature 160 kDa protein and confirm the ER retention by the prominent expression of the immature 120 kDa protein. The FLAG tag of 25 amino acids is not expressed in Non-transduced HepG2 control cells and not detected in mock transduced HepG2. Alpha tubulin represents the loading control. (B(ii)). The fold change of Alpha tubulin normalized mature and immature forms of FLAG-tagged LDLR is represented as mean + SD. n = 3. Unpaired students’ t-test, two-tailed, P < 0.05 = *, P < 0.01 = **