hDF cells were collected and washed with cold PBS (2.7 mM KCl, 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 138 mM NaCl [pH 7.4]). Total proteins were solubilized in lysis buffer (20 mM Tris–HCl [pH 7.4], 1 mM ethylenediaminetetraacetic acid (EDTA), 0.4 mM NaF, 0.04 mM Na3VO, 1% NP40, protease inhibitors) and sonicated in ice. Extraction of separate cytoplasmic and nuclear protein fractions was performed with NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by SDS-PAGE and transferred on PVDF membrane (Millipore, Bedford, Massachusetts, USA). The level of expression of TMEM175, LC3I/II, p62, LAMP1, TFEB, GAPDH, and PARP1 was determined by immunoblot analysis using anti-LAMP1 (ab25630, Abcam, Cambridge, UK, 1:1000), anti-LC3B (2775, Cell Signaling, Danvers, Massachusetts, USA, 1:1000/), anti-TMEM175 (19,925–1-AP, Proteintech, Manchester, UK, 1:1000) and anti-p62/SQSTM1 (P0067, Sigma-Aldrich, St. Louis, MO, USA, 1:15,000), anti-TFEB (A303-673A, Bethyl Laboratories (Waltham, MA, USA), 1:1000), anti-PARP1 (66,520-1Ig, Proteintech, Manchester, UK, 1:20,000), and anti-GAPDH (sc-32233, Santa Cruz Biotechnology (Dallas, Texas USA), 1:1000). Cytoplasmic proteins were normalized to GAPDH, whereas nuclear proteins were normalized to PARP1.
Immunoprecipitation (1 mg total lysate) was performed using the anti-Akt antibodies (Immunological Sciences, Cat. N. MAB-94320) complexed to protein G-Sepharose (Invitrogen, Cat. N. 101,243). The immunoprecipitated proteins were resolved on 10% SDS–PAGE, transferred for 2 h at room temperature on PVDF membrane, and detected by immunoblotting with GFP (1:1000) (Synaptic System, Cat. N. 132 002) and Akt (1:1000). HRP-conjugated secondary antibodies (Immunological Sciences, Cat. N. IS20402) were used at 1:5000 dilution. Protein bands were detected by ECL and visualized by Quantity One software (Bio-Rad Laboratories).
The mean standard deviations of 3 independent experiments were analyzed as multiple datasets with ANOVA test for multiple comparisons or with Student’s t-test. Data were plotted as histogram representation. A value of p < 0.05 was considered statistically significant.
Immunoprecipitation (1 mg total lysate) was performed using the anti-Akt antibodies (Immunological Sciences, Cat. N. MAB-94320) complexed to protein G-Sepharose (Invitrogen, Cat. N. 101,243). The immunoprecipitated proteins were resolved on 10% SDS–PAGE, transferred for 2 h at room temperature on PVDF membrane, and detected by immunoblotting with GFP (1:1000) (Synaptic System, Cat. N. 132 002) and Akt (1:1000). HRP-conjugated secondary antibodies (Immunological Sciences, Cat. N. IS20402) were used at 1:5000 dilution. Protein bands were detected by ECL and visualized by Quantity One software (Bio-Rad Laboratories).
The mean standard deviations of 3 independent experiments were analyzed as multiple datasets with ANOVA test for multiple comparisons or with Student’s t-test. Data were plotted as histogram representation. A value of p < 0.05 was considered statistically significant.
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