Journal: Autophagy

Article Title: Loss of RUBCN/rubicon in adipocytes mediates the upregulation of autophagy to promote the fasting response

doi: 10.1080/15548627.2022.2047341

Figure Lengend Snippet: RUBCN is significantly decreased in an autophagy-dependent manner during fasting. (A) Immunoblotting to detect the indicated proteins on day 10 in 3T3-L1 cells that were starved and cultured with or without 125 nM Baf A1 for the indicated times (n = 3). Data were analyzed using one-way ANOVA followed by Dunnett’s test. (B) Immunoblotting to detect the indicated proteins in wild-type eWAT depots explanted in DMEM, treated with or without 20 mM ammonium chloride and 200 μM leupeptin for 2 h (n = 3). Data were analyzed using a two-tailed Student’s t-test. (C) Immunoblotting to detect the indicated proteins in HeLa cells that were treated with 125 nM Baf A1 for the indicated times. (D) Representative immunocytochemistry images to detect MAP1LC3 and Ubiquitin in HEK293T cells stably expressing GFP-RUBCN. Cells were cultured in CM or EBSS with or without 125 nM Baf A1 for 8 h. Scale bars: 30 μm (n = 4). (E) Immunoblotting to detect the indicated proteins in wild-type or PIK3C3 knockout HeLa cells. (F) Immunoblotting to detect the indicated proteins in wild-type or Becn1 knockout MEFs. (G) Immunoblotting to detect the indicated proteins in HeLa cells that were treated with 250 nM Torin-1 for the indicated times. (H) Immunoblotting to detect the indicated proteins in wild-type or ATG7 knockout HEK293T cells that were treated with 250 nM Torin-1 for the indicated times (n = 3). Data were analyzed using one-way ANOVA followed by Dunnett’s test. Quantification of the data is shown on the graphs to the right of each blot. All data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and N.S., not significant.

Article Snippet: Reagents and antibodies The following antibodies were used for western blotting at the indicated dilutions: rabbit monoclonal anti-RUBCN/rubicon (Cell Signaling Technology [CST], 8465; 1:1000), rabbit polyclonal anti-ATG12 (CST, 2011; 1:2000), rabbit polyclonal anti-PIK3C3/VPS34 [ 22 ] (1:3000), mouse monoclonal anti-PIK3R4/VPS15 (Abnova, H00030849-M03; 1:2000), mouse monoclonal anti-BECN1/Beclin 1 (BD, 612,112; 1:2000), rabbit polyclonal anti-MAP1LC3 (MBL, PM036; 1:2000), rabbit monoclonal anti-MAP1LC3A (CST, 4599; 1:2000), rabbit polyclonal anti-MAP1LC3A/B (CST, 4108; 1:2000), rabbit monoclonal anti-MAP1LC3C (CST, 14,736; 1:2000), rabbit polyclonal anti-GABARAP (MBL, PM037; 1:2000), rabbit polyclonal anti-GABARAPL1 (abcam, ab86497; 1:2000), rabbit polyclonal anti-GABARAPL2 (MBL, PM038, 1:2000), rabbit polyclonal anti-SQSTM1/p62 (MBL, PM045; 1:5000), rabbit monoclonal anti-TAX1BP1 (CST, 5105, 1:2000), rabbit monoclonal anti-NBR1 (CST, 9891; 1:2000), rabbit monoclonal anti-OPTN/optineurin (CST, 58,981; 1:2000), rabbit monoclonal anti-CALCOCO2/NDP52 (CST, 60,732; 1:2000), rabbit polyclonal anti-ULK1 (Santa Cruz Biotechnology, sc-33,182; 1:2000), rabbit polyclonal anti-phospho-ULK1 (S757; CST, 6888; 1:2000), rabbit polyclonal anti-RPS6KB1/S6K1 (CST, 9202; 1:2000), rabbit polyclonal anti-phospho-RPS6KB1/S6K1 (T389; CST, 9205; 1:2000), rabbit polyclonal anti-AKT (CST, 9272; 1:2000), rabbit polyclonal anti-phospho-AKT (S473; CST, 9271; 1:2000), rabbit polyclonal anti-LIPE/HSL (CST, 4107; 1:2000), rabbit polyclonal anti-phospho-LIPE/HSL (S660) (CST, 45,804; 1:2000), rat monoclonal anti-ADIPOQ/adiponectin (R&D Systems, MAB1119; 1:2000), mouse monoclonal anti-FABP4 (Santa Cruz Biotechnology, sc-271,529; 1:2000), mouse monoclonal anti-PPARG (Santa Cruz Biotechnology, sc-7273; 1:2000), rabbit monoclonal anti-NCOA1/SRC-1 (CST, 2191; 1:2000), rabbit polyclonal anti-NCOA2/TIF2 (Bethyl Laboratories, A300-346A; 1:2000), mouse monoclonal antiTUBA4A/α-tubulin (Sigma-Aldrich, T5168; 1:25,000), mouse monoclonal anti-ACTB/β-actin (MBL, M177-3; 1:25,000), rabbit monoclonal anti-GAPDH (CST, 2118; 1:25,000), goat monoclonal anti-LMNB1/lamin B1 (Santa Cruz Biotechnology, sc-6217; 1/1000), HRP-conjugated goat anti–rabbit IgG (Jackson ImmunoResearch, 111–035-003; 1:2000), HRP-conjugated goat anti–rat IgG (Jackson ImmunoResearch, 112–035-003; 1:2000), and HRP-conjugated goat anti–mouse IgG (Jackson ImmunoResearch, 115–035-003; 1:2000).

Techniques: Western Blot, Cell Culture, Two Tailed Test, Immunocytochemistry, Stable Transfection, Expressing, Knock-Out

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Synaptic proteins in human cerebral organoids and neurons. a Western blots of the total lysate (h) and synaptosomes (syn) from hCOs using synaptophysin (SYP), sintaxin (STX), synaptotagmin 1/2 (SYT), and β-actin (ACTB) antibodies. Molecular weight in kDalton (kDa) is indicated on the left. Enrichment of b synaptophysin, c syntaxin, and d synaptotagmin 1/2 protein level, normalized on β-actin, in the synaptosomes (syn) compared to the homogenate (h, dotted line) of hCOs at 2, 3, 5, 7, 11, and 14 months (m). e Western blot of the synaptosomes from hCOs using cystatin B (CSTB) and ACTB antibodies. Molecular weight in kDa is indicated on the left. f CSTB protein level, normalized on β-actin, in synaptosomes of hCOs. m, months. Each time point is a pool of 20–40 hCOs. g Micrographs showing doublecortin (DCX, red), cystatin B (CSTB, white), and synaptophysin (SYP, green) in 10-week neurons from NPCs. DAPI (blu) stained nuclei. The arrowheads in the enlarged images of the boxed area indicate the colocalization of SYP and CSTB. Scale bar in each panel

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Western Blot, Molecular Weight, Staining

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: CSTB is secreted through extracellular vesicles from rodent synaptosomes. a Western blots of rat brain synaptosomes and their secreted fraction (secretome), using synaptophysin (SYP), cystatin B (CSTB), CD81, and β-actin (ACTB) antibodies. Lane 1: synaptosomes; lane 2: secreted soluble fraction; lane 3: secreted extracellular vesicles (EVs) fraction. Molecular weight is indicated on the left (kDa). The image is representative of experiments performed on samples from n = 3 rats. b Table showing the presence/absence of each protein in each fraction, as obtained from n = 3 rats. Row 1: synaptosomes; row 2: secreted soluble fraction; row 3: secreted extracellular vesicles fraction (EVs). Dark gray box indicates presence, white box indicates absence. c Micrographs of synaptosomes isolated from E18 mouse brain ( n = 5) identified by synaptophysin (SYP, green) immunostaining; CD81-positive (red, upper panels) and -negative (lower panels) synaptosomes. d Micrographs of synaptosomes isolated from E18 mouse brain identified by synaptophysin (SYP, green) immunostaining; CSTB-positive (red, upper panels) and -negative (lower panels) synaptosomes. e Micrographs showing CD81-positive (green) EVs secreted from E18 mouse brain synaptosomes; CSTB-positive (red, upper panels) and -negative (lower panels) EVs. Scale bar in each panel

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Western Blot, Molecular Weight, Isolation, Immunostaining

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Distribution and release of EVs proteins and CSTB from hCOs synaptosomes. a Western blots of the total lysate (h) and synaptosomes (syn) from 2, 3, 5, 7, 11, and 14-month-old hCOs using antibodies for the EVs markers CD9, CD81, and CD82 and β-actin (ACTB). b Quantification of the expression levels of CD9, CD81, and CD82, normalized on the corresponding β-actin. c – f Western blots of proteins from the synaptosomal fractions upon incubation in depolarizing media (d-syn) and their secreted extracellular vesicles (EVs) and soluble fraction (SF), using CD81, cystatin B (CSTB), and synaptophysin (SYP) antibodies. Synaptosomal samples from hCOs at different maturation stages: c 3 months, d 5 months, e 8 months, and f 9 months. Each time point is a pool of 20–40 hCOs. Molecular weight of each protein is indicated on the left (kDa). m, months

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Western Blot, Expressing, Incubation, Molecular Weight

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Pathological low expression levels of CSTB results in synaptic impairment in EPM1 hCOs. Western blots of a total lysate and b synaptosomal fractions from controls and EPM1 hCOs at different developmental stages using cystatin B (CSTB) and β-actin (ACTB) antibodies. Western blots of c total lysate and d synaptosomal fractions from controls and EPM1 hCOs using eukariotic Initiation Factor 4G2 (eIF4G2) and ACTB antibodies. Western blots (upper panels) and relative quantifications (lower panels). e Western blots of synaptosomes from controls and EPM1 hCOs at different developmental stages, using synaptophysin (SYP), sintaxin (STX), synaptotagmin 1/2 (SYT), and ACTB antibodies and relative quantifications. Molecular weight of each protein is indicated on the left (kDa). Data are presented as mean ± SD from n = 2 pools of 20–40 organoids. Controls: C1, C2; patients: E1, E2; m, months

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Expressing, Western Blot, Molecular Weight

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Altered expression levels of EVs markers in synaptosomes from EPM1 hCOs. a Western blots of synaptosomal proteins from controls and EPM1 hCOs at different developmental stages using CD9, CD81, and β-actin (ACTB) antibodies. Quantifications of the expression levels of b CD9 and c CD81 normalized on ACTB. Data are presented as mean ± SD from n = 2 pools of 20–40 organoids. Controls: C1, C2; patients: E1, E2; m, months. d Venn diagram of the proteins identified by mass spectrometry enriched in the synaptosomal fraction (syn) from 1.5-m control hCOs (CTRL) (76 proteins, syn/h ≥ 1.2) and depleted in EPM1 synaptosomes (31 proteins, syn/h ≤ 0.8). e Gene Ontology analysis of the 31 proteins depleted in EPM1 synaptosomes, using String database. h, homogenate

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Expressing, Western Blot, Mass Spectrometry, Control