Figure S7.
Validation of shRNA constructs. (A) Validation of shRNA clones to knockdown SNX17 and SNX27 in rat cortical neurons. Neurons were infected with lentiviruses carrying either control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002, Millipore Sigma), SNX17-shRNA (TRCN0000190340, Millipore Sigma) or SNX27-shRNA (TRCN0000253473, Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (B) SNX17 protein levels were quantified in neurons infected with lentiviruses carrying SNX17- or SNX27-shRNAs and normalized to the protein levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; SNX17-shRNA: 32.2 ± 3.52%, SNX27-shRNA: 100.3 ± 5.85%. N = 3 independent experiments. Statistical significance was determined using ANOVA with Tukey’s post hoc comparisons, ****P < 0.001. Error bars are SEM. (C) The protein levels of SNX27 were quantified in neurons infected with lentiviruses carrying SNX17- or SNX27-shRNAs and normalized to the protein levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; SNX17-shRNA: 102.3 ± 9.21%, SNX27-shRNA: 37.96 ± 0.29%. N = 3 independent experiments. Statistical significance was determined using ANOVA with Tukey’s post hoc comparisons, ***P < 0.005. Error bars are SEM. (D) Validation of an shRNA clone to knockdown rat VPS26B. Rat cortical neurons were infected with lentiviruses carrying either control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002; Millipore Sigma) or VPS26B-shRNA (TRCN0000306336; Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (E) HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or SNX27-shRNA in the absence or presence of eGFP, GFP-SNX27, or shRNA-resistant GFP-SNX27 (SNX27-R), as indicated. Five days after infection, cells were treated with 1 μg/ml of doxycycline to promote the expression of the eGFP-tagged constructs. 24 h later, extracts were collected and analyzed by western blot. Source data are available for this figure: SourceData FS7.

Validation of shRNA constructs. (A) Validation of shRNA clones to knockdown SNX17 and SNX27 in rat cortical neurons. Neurons were infected with lentiviruses carrying either control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002, Millipore Sigma), SNX17-shRNA (TRCN0000190340, Millipore Sigma) or SNX27-shRNA (TRCN0000253473, Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (B) SNX17 protein levels were quantified in neurons infected with lentiviruses carrying SNX17- or SNX27-shRNAs and normalized to the protein levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; SNX17-shRNA: 32.2 ± 3.52%, SNX27-shRNA: 100.3 ± 5.85%. N = 3 independent experiments. Statistical significance was determined using ANOVA with Tukey’s post hoc comparisons, ****P < 0.001. Error bars are SEM. (C) The protein levels of SNX27 were quantified in neurons infected with lentiviruses carrying SNX17- or SNX27-shRNAs and normalized to the protein levels in control-shRNA–infected neurons. ctrl-shRNA: 100%; SNX17-shRNA: 102.3 ± 9.21%, SNX27-shRNA: 37.96 ± 0.29%. N = 3 independent experiments. Statistical significance was determined using ANOVA with Tukey’s post hoc comparisons, ***P < 0.005. Error bars are SEM. (D) Validation of an shRNA clone to knockdown rat VPS26B. Rat cortical neurons were infected with lentiviruses carrying either control-shRNA (pLKO.1 scrambled nontarget shRNA SHC002; Millipore Sigma) or VPS26B-shRNA (TRCN0000306336; Millipore Sigma) at an MOI of 2. At 6 days after infection, cell extracts were collected and analyzed by western blot. GAPDH was used as a loading control. (E) HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or SNX27-shRNA in the absence or presence of eGFP, GFP-SNX27, or shRNA-resistant GFP-SNX27 (SNX27-R), as indicated. Five days after infection, cells were treated with 1 μg/ml of doxycycline to promote the expression of the eGFP-tagged constructs. 24 h later, extracts were collected and analyzed by western blot. Source data are available for this figure: SourceData FS7.

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