Fig. 5

(A) After 48 h of transfection with pCDNA6.0-His-Gag plasmid in HEK293T cells, real-time PCR was performed to detect changes in Rab5a mRNA levels. (B, C) Western blot analysis revealed changes in Rab5a protein levels following 48 h of transfection with pCDNA6.0-His-Gag plasmid in HEK293T cells (**p < 0.01, ***p < 0.001). (D) HEK293T cells were transfected with pcDNA6.0 (empty vector), pCDNA6.0-His-Gag plasmid, Rab5a expression plasmid, shRNA targeting Rab5a, or cotransfected with pCDNA6.0-His-Gag plasmid and shRNA targeting Rab5a. Cell lysates were collected, proteins extracted, and Western blotting was used to detect changes in p62, LC3 II/I, and Tom20. (E-G) Statistical analysis of p62, Tom20, and LC3 II/I protein levels (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05). (H) Following the same transfection as in (D), cells were collected for mitochondrial isolation. Proteins in mitochondria were extracted, and Western blot analysis was performed to assess changes in p62 and LC3 II/I in cytoplasm and mitochondria. (I-J) Statistical analysis of changes in p62 and LC3 II/I in cytoplasmic mitochondria. (Student’s t-test, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05)