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: Analysis of spinal cords from 3-month-old wild-type (wt) and Stam1 KO mice. Quantitation and representative immunoblots of (A) ESCRT proteins STAM1 (p = 0.0001), HGS (p = 0.0352), TSG101 (p = 0.2422), and VPS36 (p = 0.6694) and (B) endosomal proteins APPL1 (p = 0.6239), ALIX (p = 0.9725), CLTC (p = 0.1518), EEA1 (p = 1.0), LAMP2A (p = 0.1688), M6PR (p = 0.3739), MAP1LC3A (p = 0.3951), RAB3 (p = 0.7899), RAB5 (p = 0.0926), RAB7 (p = 0.3506), RAB11 (p = 0.2578) and RAB35 (p = 0.953). ACTB was used as a loading control. (C) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for EEA1 and DAPI. (D) Cumulative frequency plot showing the size distribution of EEA1 puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (E) size (p < 0.00001), (F) fluorescence intensity (p = 0.2293), and (G) number (p = 0.5026) of EEA1 puncta in 3-month-old wt and Stam1 KO mice. (H) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7429). (I) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for LAMP2A and DAPI. (J) Cumulative frequency plot showing the size distribution of LAMP2A puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (K) size (p < 0.00001), (L) fluorescence intensity (p = 0.8022), and (M) number (p = 0.777) of LAMP2A puncta in 3-month-old wt and Stam1 KO mice. (N) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). *p < 0.05, ****p < 0.0001.

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: Quantitation Assay, Western Blot, Control, Staining, Fluorescence

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: 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: STAM1 deletion causes reduced expression of VTI1A and VAMP2. (A) Quantitation and representative immunoblots showing levels of SNAP25 (p = 0.7208), SV2A (p = 0.6292), SYN1 (p = 0.9335), SYP (p = 0.4278), SYT1 (p = 0.7787), STX1A (p = 0.2887), STX1B (p = 0.1477), STX6 (p = 0.7953), VAMP1 (p = 0.1282), VAMP2 (p = 0.0247) and VTI1A (p = 0.0049) in the spinal cords of 3-month-old wild-type (wt) and Stam1 KO mice. When multiple known isoforms of a protein were detected, an asterisk was placed next to immunoblot to indicate the protein bands that were quantitated. ACTB was used as a loading control. (B) Representative L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for VTI1A and DAPI. (C) Cumulative frequency plot showing the size distribution of VTI1A puncta in motor neurons of 3-month-old wt and Stam1 KO mice. The Kolmogorov-Smirnov test was used to determine significance. Quantitation of average (D) size (p < 0.0001), (E) fluorescence intensity (p = 0.0004) and (F) number (p = 0.4337) of VTI1A puncta in L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice. (G) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). (H) Immunoblot analysis of proteins that co-immunoprecipitated with STAM1 in spinal cord lysates from 1-month-old wild-type mice. Lanes represent input lysate, proteins that did not bind to the beads, and proteins that were captured by the STAM1 antibody bead complex. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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: Expressing, Quantitation Assay, Western Blot, Control, Staining, Fluorescence, Immunoprecipitation

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: STAM1 deletion causes reduced expression of VTI1A and VAMP2. (A) Quantitation and representative immunoblots showing levels of SNAP25 (p = 0.7208), SV2A (p = 0.6292), SYN1 (p = 0.9335), SYP (p = 0.4278), SYT1 (p = 0.7787), STX1A (p = 0.2887), STX1B (p = 0.1477), STX6 (p = 0.7953), VAMP1 (p = 0.1282), VAMP2 (p = 0.0247) and VTI1A (p = 0.0049) in the spinal cords of 3-month-old wild-type (wt) and Stam1 KO mice. When multiple known isoforms of a protein were detected, an asterisk was placed next to immunoblot to indicate the protein bands that were quantitated. ACTB was used as a loading control. (B) Representative L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for VTI1A and DAPI. (C) Cumulative frequency plot showing the size distribution of VTI1A puncta in motor neurons of 3-month-old wt and Stam1 KO mice. The Kolmogorov-Smirnov test was used to determine significance. Quantitation of average (D) size (p < 0.0001), (E) fluorescence intensity (p = 0.0004) and (F) number (p = 0.4337) of VTI1A puncta in L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice. (G) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). (H) Immunoblot analysis of proteins that co-immunoprecipitated with STAM1 in spinal cord lysates from 1-month-old wild-type mice. Lanes represent input lysate, proteins that did not bind to the beads, and proteins that were captured by the STAM1 antibody bead complex. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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: Expressing, Quantitation Assay, Western Blot, Control, Staining, Fluorescence, Immunoprecipitation

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: Analysis of spinal cords from 3-month-old wild-type (wt) and Stam1 KO mice. Quantitation and representative immunoblots of (A) ESCRT proteins STAM1 (p = 0.0001), HGS (p = 0.0352), TSG101 (p = 0.2422), and VPS36 (p = 0.6694) and (B) endosomal proteins APPL1 (p = 0.6239), ALIX (p = 0.9725), CLTC (p = 0.1518), EEA1 (p = 1.0), LAMP2A (p = 0.1688), M6PR (p = 0.3739), MAP1LC3A (p = 0.3951), RAB3 (p = 0.7899), RAB5 (p = 0.0926), RAB7 (p = 0.3506), RAB11 (p = 0.2578) and RAB35 (p = 0.953). ACTB was used as a loading control. (C) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for EEA1 and DAPI. (D) Cumulative frequency plot showing the size distribution of EEA1 puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (E) size (p < 0.00001), (F) fluorescence intensity (p = 0.2293), and (G) number (p = 0.5026) of EEA1 puncta in 3-month-old wt and Stam1 KO mice. (H) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7429). (I) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for LAMP2A and DAPI. (J) Cumulative frequency plot showing the size distribution of LAMP2A puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (K) size (p < 0.00001), (L) fluorescence intensity (p = 0.8022), and (M) number (p = 0.777) of LAMP2A puncta in 3-month-old wt and Stam1 KO mice. (N) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). *p < 0.05, ****p < 0.0001.

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: Quantitation Assay, Western Blot, Control, Staining, Fluorescence

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: Pathological analysis of Stam1 KO spinal cords. (A) Representative images of multi-plex fluorescent in situ hybridization for ( ii.) choline acetyltransferase ( Chat ), ( iii. ) Stam1 , and ( iv .) Hgs mRNA in sections co-stained with ( i. ) DAPI. The boxed area is zoomed in to show expression of ( vi .) Chat , ( vii. ) Stam1 and ( viii. ) Hgs as well as ( v. ) DAPI in motor neurons. ( ix. ) Merged images of Chat , Stam1 , Hgs and DAPI. Relative expression levels of (B) Stam1 and (C) Hgs in different cell types in the mouse brain and spinal cord (from mousebrain.org ). (D–G) Analysis of lumbar 4/5 spinal segment pathology in Stam1 KO mice. (D) Representative L4/5 spinal cord sections and quantitation of motor neurons (p = 0.2802) from spinal cords of 3-month-old wt and Stam1 KO mice containing the Thy1-Yfp transgene. Spinal cords from 3-month-old wt and Stam1 KO mice stained for DAPI and either (E) cleaved caspase 3 (CC3), (F) GFAP (p = 0.017), or (G) IBA1 (p = 0.0929). Since no CC3 positive cells were identified in either the 3-month-old wt or Stam1 KO spinal cords, brain sections from embryonic day 17 (E17) wt mice were included as a positive control for CC3 staining. *p < 0.05.

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: In Situ Hybridization, Staining, Expressing, Quantitation Assay, Positive Control

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: 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: 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: Characterization of Stam1 KO mice. (A) Survival curves for wild-type (wt) and Stam1 KO mice. (B) Body mass (1 month p = 0.6502, 3 month p = 0.0027), (C) gastrocnemius mass (1 month p = 0.147, 3 month p = 0.0001), (D) percent time ambulatory in open field (1 month p = 0.0057, 3 month p = 0.0224), (E) distance traveled in open field (1 month p = 0.0117, 3 month p = 0.0274), (F) average velocity in open field (1 month p = 0.4394, 3 month p = 0.9742), (G) latency to fall (1 month p = 0.1771, 3 month p = 0.0001), and (H) fore-limb grip strength in grams force (gf) (1 month p = 0.3749, 3 month p = 0.0045) of wt and Stam1 KO mice. (I) Mechanical allodynia determined by von Frey assay (p = 0.0002) of 3-month-old wt and Stam1 KO mice. Data are shown as mean ± SEM, n = 6 mice per genotype for A-H and 4 for I. Open boxes indicate wt mice and grey boxes indicate Stam1 KO mice. Filled circles indicate female mice and filled squares represent male mice. *p < 0.05, **p < 0.01, ***p < 0.001.

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:

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: Analysis of spinal cords from 3-month-old wild-type (wt) and Stam1 KO mice. Quantitation and representative immunoblots of (A) ESCRT proteins STAM1 (p = 0.0001), HGS (p = 0.0352), TSG101 (p = 0.2422), and VPS36 (p = 0.6694) and (B) endosomal proteins APPL1 (p = 0.6239), ALIX (p = 0.9725), CLTC (p = 0.1518), EEA1 (p = 1.0), LAMP2A (p = 0.1688), M6PR (p = 0.3739), MAP1LC3A (p = 0.3951), RAB3 (p = 0.7899), RAB5 (p = 0.0926), RAB7 (p = 0.3506), RAB11 (p = 0.2578) and RAB35 (p = 0.953). ACTB was used as a loading control. (C) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for EEA1 and DAPI. (D) Cumulative frequency plot showing the size distribution of EEA1 puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (E) size (p < 0.00001), (F) fluorescence intensity (p = 0.2293), and (G) number (p = 0.5026) of EEA1 puncta in 3-month-old wt and Stam1 KO mice. (H) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7429). (I) L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for LAMP2A and DAPI. (J) Cumulative frequency plot showing the size distribution of LAMP2A puncta in motor neurons from 3-month-old wt and Stam1 KO L4/5 spinal cord sections . The Kolmogorov-Smirnov test was used to determine significance. Average (K) size (p < 0.00001), (L) fluorescence intensity (p = 0.8022), and (M) number (p = 0.777) of LAMP2A puncta in 3-month-old wt and Stam1 KO mice. (N) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). *p < 0.05, ****p < 0.0001.

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: Quantitation Assay, Western Blot, Control, Staining, Fluorescence

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: Pathological analysis of Stam1 KO spinal cords. (A) Representative images of multi-plex fluorescent in situ hybridization for ( ii.) choline acetyltransferase ( Chat ), ( iii. ) Stam1 , and ( iv .) Hgs mRNA in sections co-stained with ( i. ) DAPI. The boxed area is zoomed in to show expression of ( vi .) Chat , ( vii. ) Stam1 and ( viii. ) Hgs as well as ( v. ) DAPI in motor neurons. ( ix. ) Merged images of Chat , Stam1 , Hgs and DAPI. Relative expression levels of (B) Stam1 and (C) Hgs in different cell types in the mouse brain and spinal cord (from mousebrain.org ). (D–G) Analysis of lumbar 4/5 spinal segment pathology in Stam1 KO mice. (D) Representative L4/5 spinal cord sections and quantitation of motor neurons (p = 0.2802) from spinal cords of 3-month-old wt and Stam1 KO mice containing the Thy1-Yfp transgene. Spinal cords from 3-month-old wt and Stam1 KO mice stained for DAPI and either (E) cleaved caspase 3 (CC3), (F) GFAP (p = 0.017), or (G) IBA1 (p = 0.0929). Since no CC3 positive cells were identified in either the 3-month-old wt or Stam1 KO spinal cords, brain sections from embryonic day 17 (E17) wt mice were included as a positive control for CC3 staining. *p < 0.05.

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: In Situ Hybridization, Staining, Expressing, Quantitation Assay, Positive Control

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: 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: STAM1 deficiency does not affect peripheral nerve myelination. (A) Representative electron micrographs of sciatic nerves from 3-month-old wild-type (wt) and Stam1 KO mice. Quantitation showing (B) the percent of sciatic nerve axons >1 μm in diameter that were myelinated (p = 0.9286), (C) the ratio of axon diameter to total nerve fiber thickness (g-ratio) (p = 0.1205), and (D) axon density (p = 0.9541) in sciatic nerves from wt and Stam1 KO mice.

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: Quantitation Assay

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: STAM1 deletion causes NMJ and muscle defects in Stam1 KO mice. (A) Representative muscle fibers from tibialis anterior muscles of 3-month-old wild-type (wt) and Stam1 KO mice containing the Thy1-Yfp transgene were stained with TRITC-α-bungarotoxin (red) to label the postsynaptic AChRs. The presynaptic axons and terminals were visualized by fluorescence from the Thy1-Yfp transgene (green). # represents swollen terminals and * represents synaptic terminal sproutings in the Stam1 KO mice. Quantitation of NMJs from (B) 1-month-old or (C) 3-month-old wt and Stam1 KO mice that showed denervation (1 month p = 0.1483, 3 month p = 0.0007), swellings (1 month p = 0.001, 3 month p = 0.0012) or terminal sproutings (1 month p = 0.0068, 3 month p = 0.0005). Quantitation of NMJ area from (D) 1-month-old (p = 0.7007) and (E) 3-month-old (p = 0.03) wt and Stam1 KO mice. Quantitation of cross-sectional area from gastrocnemius muscles of (F) 1-month-old (p = 0.0659) and (G) 3-month-old (p = 0.0011) wt and Stam1 KO mice. qPCR analysis of AChR expression in gastrocnemius muscles of (H) 1-month-old and (I) 3-month-old wt or Stam1 KO mice (AChR-α: 1 month p = 0.066, 3 month p = 0.2428; AChR-β: 1 month p = 0.055, 3 month p = 0.4802; AChR-δ: 1 month p = 0.0351, 3 month p = 0.00364; AChR-ε: 1 month p = 0.2638, 3 month p = 0.0036; and AChR-γ: 1 month p = 0.0788, 3 month p = 0.0002). Open boxes indicate wt mice and grey boxes indicate Stam1 KO mice. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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: Muscles, Staining, Fluorescence, Quantitation Assay, Expressing

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: STAM1 deletion causes reduced expression of VTI1A and VAMP2. (A) Quantitation and representative immunoblots showing levels of SNAP25 (p = 0.7208), SV2A (p = 0.6292), SYN1 (p = 0.9335), SYP (p = 0.4278), SYT1 (p = 0.7787), STX1A (p = 0.2887), STX1B (p = 0.1477), STX6 (p = 0.7953), VAMP1 (p = 0.1282), VAMP2 (p = 0.0247) and VTI1A (p = 0.0049) in the spinal cords of 3-month-old wild-type (wt) and Stam1 KO mice. When multiple known isoforms of a protein were detected, an asterisk was placed next to immunoblot to indicate the protein bands that were quantitated. ACTB was used as a loading control. (B) Representative L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for VTI1A and DAPI. (C) Cumulative frequency plot showing the size distribution of VTI1A puncta in motor neurons of 3-month-old wt and Stam1 KO mice. The Kolmogorov-Smirnov test was used to determine significance. Quantitation of average (D) size (p < 0.0001), (E) fluorescence intensity (p = 0.0004) and (F) number (p = 0.4337) of VTI1A puncta in L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice. (G) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). (H) Immunoblot analysis of proteins that co-immunoprecipitated with STAM1 in spinal cord lysates from 1-month-old wild-type mice. Lanes represent input lysate, proteins that did not bind to the beads, and proteins that were captured by the STAM1 antibody bead complex. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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: Expressing, Quantitation Assay, Western Blot, Control, Staining, Fluorescence, Immunoprecipitation

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: STAM1 deletion causes reduced expression of VTI1A and VAMP2. (A) Quantitation and representative immunoblots showing levels of SNAP25 (p = 0.7208), SV2A (p = 0.6292), SYN1 (p = 0.9335), SYP (p = 0.4278), SYT1 (p = 0.7787), STX1A (p = 0.2887), STX1B (p = 0.1477), STX6 (p = 0.7953), VAMP1 (p = 0.1282), VAMP2 (p = 0.0247) and VTI1A (p = 0.0049) in the spinal cords of 3-month-old wild-type (wt) and Stam1 KO mice. When multiple known isoforms of a protein were detected, an asterisk was placed next to immunoblot to indicate the protein bands that were quantitated. ACTB was used as a loading control. (B) Representative L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice stained for VTI1A and DAPI. (C) Cumulative frequency plot showing the size distribution of VTI1A puncta in motor neurons of 3-month-old wt and Stam1 KO mice. The Kolmogorov-Smirnov test was used to determine significance. Quantitation of average (D) size (p < 0.0001), (E) fluorescence intensity (p = 0.0004) and (F) number (p = 0.4337) of VTI1A puncta in L4/5 spinal cord segments from 3-month-old wt and Stam1 KO mice. (G) Average area of motor neurons in 3-month-old wt and Stam1 KO mice (p = 0.7609). (H) Immunoblot analysis of proteins that co-immunoprecipitated with STAM1 in spinal cord lysates from 1-month-old wild-type mice. Lanes represent input lysate, proteins that did not bind to the beads, and proteins that were captured by the STAM1 antibody bead complex. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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: Expressing, Quantitation Assay, Western Blot, Control, Staining, Fluorescence, Immunoprecipitation