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. 2017 Jan 23;26(4):729–741. doi: 10.1093/hmg/ddw413

Figure 4.

Figure 4.

Rab8a mediates retromer cargo CI-M6PR trafficking, secretory autophagy and Golgi-derived vesicle secretion defects upon TMEM230 knockdown. (A) TMEM230 depletion reduces Rab8a but not Rab7a protein levels. HEK293-FT cells were transfected either with control Scr-RNAi or TMEM230-RNAi. Two days later, transfected cells were analysed with immunoblotting using TMEM230, Rab8a, Rab7a and tubulin antibodies. Immunoblot band intensities were normalized to tubulin, and compared to Scr-RNAi. Graphs show normalized Rab8a immunoblot intensity in cell lysates. Data represent mean ± SEM; N = 4, two-tailed t-test, ***=P < 0.001 compared to Scr-RNAi. (B-I) Rab8a depletion reduces steady state levels of CI-M6PR retromer cargo levels and increases BafA1-induced intracellular accumulation of autophagic cargo (p62 and LC3-II) and Golgi-derived vesicle cargo (immature lysosomal hydrolases iCat-D and iHEX-B), and partially affects lysosomal cargo levels (mature lysosomal hydrolases mCat-D and mHEX-B). HEK293-FT cells were transfected with either Scr-RNAi or Rab8a-RNAi. Two days later, cells were treated with 300 nM Baf-A1 for the indicated time. Extracellular media fractions were prepared as described in Experimental Procedures. Cells were lysed with 2X SDS-sample buffer and cell lysates were analysed by immunoblotting with indicated antibodies. (B) Representative immunoblot data from cell lysates. Band intensities were normalized to total tubulin, and compared to Scr-RNAi. Graphs show normalized band intensities of intracellular CI-M6PR (N = 5) (C), intracellular p62 (N = 5) (D), intracellular LC3-II (N = 5) (E), intracellular immature cathepsin-D (N = 5) (F), intracellular mature cathepsin-D (N = 5) (G), intracellular immature HEX-B (N = 5) (H) and intracellular mature HEX-B (N = 5) (I). Data represent mean ± SEM; two-tailed paired t-test, *=P < 0.05; **=P < 0.01 compared with Scr-RNAi. (J-M) Rab8a depletion inhibits Baf-A1-induced extracellular secretion of autophagic cargo (p62), Golgi-derived vesicle cargo (immature lysosomal hydrolase iHEX-B) and lysosomal cargo (mature lysosomal hydrolase mHEX-B). After Baf-A1 treatment for indicated times, harvested extracellular media fractions were analysed with immunoblotting using indicated antibodies. (J) Representative immunoblot data from extracellular media fractions. Blot band intensities were normalized to total tubulin, and compared to Scr-RNAi. Graphs show normalized immunoblot band intensities of p62 from extracellular media (N = 3) (K), immature HEX-B from extracellular media (N = 4) (L), and mature HEX-B from extracellular media (N = 4) (M). Data represent mean ± SEM; two-tailed paired t-test, *=P < 0.05, compared with control Scr-RNAi.