Journal: Current Research in Neurobiology

Article Title: Analysis of the neuromuscular deficits caused by STAM1 deficiency

doi: 10.1016/j.crneur.2024.100138

Figure Lengend Snippet: Examination of ESCRT substrates and signaling pathways in spinal cords of Stam1 KO mice and controls. Quantitation and representative immunoblots of (A) the putative ESCRT substrates CXCR4 (p = 0.2268), EGFR (p = 0.3782), ERBB2 (p = 0.3127), ERBB3 (p = 0.0025), ERBB4 (p = 0.6897), GRIA1 (p = 0.1423) and NTRK2 (p = 0.685), (B) signaling molecules AKT (p = 0.6817), pAKT308 (p = 0.8087), ERK (p = 0.8911), pERK (p = 0.6793), GSK3β (p = 0.9306), pGSK3β (p = 0.5961), JNK (p = 0.1324) and pJNK (p = 0.3227), and (C) the aggregate-prone proteins PRNP (p = 0.8684), MAPT (p = 0.3193) and TARDBP (p = 0.4392) in spinal cord extracts of 3-month-old wild-type (wt) and Stam1 KO mice. When multiple known isoforms of a protein were detected, either an asterisk or bracket was placed next to the immunoblot to indicate bands that were quantitated. TUBB3 was used as a loading control. **p < 0.01.

Article Snippet: Immunoblots were probed for TSG101(cat. # sc-7964), VPS36 (cat. # sc-79930), RAB3 (cat. # sc-136050), MAPT (cat. # sc-32274), PRNP (cat. # sc-69896), and SNAP25 (cat. # sc-7538) from Santa Cruz (Dallas, TX), APPL1 (cat. # 3858), LAMP2A (cat. # 9091), MAP1LC3A (cat. # 12741), EEA1 (cat. # 3288), RAB5 (cat. # 3547), RAB7 (cat. # 9367), RAB11 (cat. # 5589), ERBB2 (cat. # 2165), ERBB3 (cat. # 12709), ERBB4 (cat. # 4795), GRIA1 (cat. # 13185), AKT (cat. # 9272), pAKT308 (cat. # 13038), ERK (cat. # 9201), pERK (cat. # 4370), JNK (cat. # 9252), pJNK (cat. # 4668), GSK3β (cat. # 9315), pGSK3β (cat. # 5585), HGS (cat. # 15087), STAM1 (cat. # 13053), CLTC (cat. # 4796), STX6 (cat. # 2869), SYN1 (cat. # 5297), VAMP1 (cat. # 13151), VAMP2 (cat. # 13508), and VTI1A (cat. # 14764) from Cell Signaling Technologies (Danvers, MA), ALIX (cat. # MA5-32773), M6PR (cat. # PA5-111123) and SYP (cat. # MA5-14532) from Thermo Fisher Scientific, CXCR4 (cat. # 11073-2-AP) from ProteinTech (Rosemount, IL), NTRK2 (cat. # AB_310445), EGFR (cat. # 06–847) from MilliporeSigma (Burlington, MA), SYT1 (cat. # 105 008), STX1A (cat. # 110 302) and STX1B (cat. # 110 402) from Synaptic Systems (Göttingen, Germany), and SV2A (cat. # SV2), ACTB (cat. # JLA20) and TUBB3 (cat. #E7) from Developmental Hybridoma Bank (Iowa City, IA).

Techniques: Protein-Protein interactions, Quantitation Assay, Western Blot, Control

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: ( A ) Non-infected PK1 cells and persistently infected PK1 cells (iS7) were immuno-stained with anti-PrP antibodies 6D11 and 5B2 (green), with anti-syntaxin-6 antibody (red) and with DAPI (blue). Förster resonance energy transfer (FRET) analysis reveals interaction in perinuclear compartments (wide arrows) and at membranes in infected cells (narrow arrows). Panels show zoomed regions of images in . ( B, C ) In vitro prion replication by protein misfolding cyclic amplification (PMCA) using Stx6 +/+ and Stx6 -/- mouse brains as substrate. PMCA reactions were seeded with RML prions from terminally ill mice and subjected to PMCA for 96 cycles over 48 hr. ( B ) Representative western blot (ICSM35) after PK digestion. Molecular weight markers are indicated on the left. ( C ) The PrP Sc signal was quantified using densitometry and normalized to the control unamplified reaction. Bar graphs each represent mean ± SD of biological replicates from three separate mice, each blotted as two technical replicates. Source data is available at https://doi.org/10.17632/yggpkrgnx8.1 .

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Infection, Staining, Förster Resonance Energy Transfer, In Vitro, Protein Misfolding Cyclic Amplification, Western Blot, Molecular Weight, Control

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: ( A, B ) PixFRET analysis of non-infected PK1 cells and persistently infected PK1 cells (iS7). Cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green), with anti-syntaxin-6 antibody (STX6, red) and with DAPI. ( C ) Schematic of binding locations of antibodies used in FRET analysis (5B2, 6D11, 8H4). Numbers represent putative epitopes. ( C ) was adapted from Figure 2B from .

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Infection, Staining, Binding Assay

Journal: eLife

Article Title: Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates

doi: 10.7554/eLife.83320

Figure Lengend Snippet: PixFRET analysis of control cells. iS7 cells were immuno-stained with anti-PrP antibodies 6D11, 8H4, or 5B2 (green) only, with anti-syntaxin-6 antibody (STX6, red) only, or with secondary antibodies only and with DAPI.

Article Snippet: Following five washes, the cells were incubated with anti-syntaxin-6 (Cell Signaling Technologies [#2869], clone C34B2, 1:300) and/or anti-PrP (BioLegend [808001] clone 6D11, 1:10,000; Santa Cruz [sc-47730], 5B2, 1:500; Sigma [P0110], clone 8H4, 1:500) in sterile-filtered 0.25× SuperBlock in PBS overnight at 4°C, followed by secondary antibodies (AlexaFluor 647-AffiniPure Goat Anti-Rabbit IgG (H+L) and/or Rhodamine Red-X (RRX) AffiniPure Goat Anti-Mouse IgG (H+L), 1:1000) and a DNA counterstain (DAPI; 1:10,000) in 0.25× SuperBlock overnight at 4°C.

Techniques: Control, Staining