Fig. 4

TMEM106B interacts with LAMP2A and regulates LAMP2A protein level. (A, B) Co-IP showing the interaction between TMEM106B and LAMP2A in HeLa cells with co-expression of FLAG-TMEM106B and HA-LAMP2A. (C) GST pull-down assays showing the interaction between GST-LAMP2A and FLAG-tagged wild-type TMEM106B. (D–F) RT-qPCR analysis showed that the mRNA level of lamp2a was not affected by tmem106bb KO in tmem106bb−/− embryos 3 dpf (n = 3 samples per group with 20 embryos in each sample), knockdown with TMEM106B siRNA or overexpression of TMEM106B in HeLa cells (n = 3 samples per group). (G, H) Western blot analysis showed that the LAMP2A protein level was significantly decreased by knockdown of TMEM106B with siRNA compared with NC siRNA in HeLa cells or increased with overexpression of TMEM106B in HeLa cells (n = 3 samples per group). (I) Pyruvate kinase activity was significantly decreased in tmem106bb−/− embryos at 3 dpf (n = 4 samples per group with 100 embryos in each sample). (J) Pyruvate kinase activity was significantly decreased in HeLa cells with knockdown of TMEM106B by siRNA compared with cells transfected with NC siRNA (n = 4 samples per group). (K) Western blot analysis showed that the FASD2 protein level was significantly increased in HeLa cells with knockdown of TMEM106B by siRNA compared with cells transfected with NC siRNA (n = 3 samples per group). *p < .05, **p < .01, ns, no significance; Data were shown as mean ± SD; Statistical analysis was performed using a Shapiro–Wilk test for samples with normal distribution and paired student's t tests with two tails.