Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A) Cebpb mRNA abundance in the ipsilateral (Ipsi) and contralateral (Contral) L3/4 DRGs (top) and C/EBPβ protein expression in the ipsilateral L3/4 DRGs (bottom) after CCI. Unilateral L3/4 DRGs from two mice were pooled together to obtain enough RNA and protein. n = 3 biological replicates (six mice) per time point. One-way analysis of variance (ANOVA) followed by Tukey post hoc test, F3,11 = 26.87 for Cebpb mRNA and F3,11 = 14.04 for C/EBPβ protein. *P < 0.05 or **P < 0.01 compared to the corresponding control group (0 day). H3, histone 3. (B) C/EBPβ protein abundance in the contralateral L3/4 DRGs after CCI. Representative Western blots and a summary of densitometric analysis are shown. n = 3 biological replicates (six mice) per time point. One-way ANOVA followed by Tukey post hoc test, F3,11 = 0.49. (C) C/EBPβ protein abundance in the ipsilateral L3/4 spinal cord after CCI. n = 3 biological replicates (three mice) per time point. One-way ANOVA followed by Tukey post hoc test, F3,11 = 1.99. (D) In situ hybridization histochemistry (ISHH) for Cebpb mRNA and immunohistochemistry of different DRG cell markers: β III tubulin, glutamine synthetase (GS), NF200, IB4, or CGRP in the DRG. n = 3 mice. Scale bar, 25 µm. (E) Distribution of Cebpb mRNA–labeled neuronal somata. Large, 31%; medium, 43%; small, 26%. (F) Neurons labeled by Cebpb mRNA in the ipsilateral L4 DRG on day 7 after CCI or sham surgery. n = 3 mice per group. **P < 0.01 compared to the corresponding sham group by two-tailed paired Student’s t test. Scale bar, 25 µm.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Quantitative Proteomics, Western Blot, In Situ Hybridization, Immunohistochemistry, Labeling, Two Tailed Test

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A) The abundance of C/EBPβ, OCT1, and mTOR after transfection of Cebpb or negative control siRNA (NC) into cultured DRG neurons. Representative Western blots and a summary of densitometric analysis are shown. n = 3 biological replicates per treatment. One-way ANOVA followed by Tukey post hoc test, F2,8 = 9.647 for C/EBPβ, F2,8 = 0.497 for OCT1, and F2,8 = 0.037 for mTOR. *P < 0.05 compared to naïve group. GAPDH, glyceraldhyde-3-phosphate dehydrogenase. (B and C) Effect of pre-microinjection of Cebpb siRNA (Si) into the ipsilateral L3/4 DRGs on basal or CCI-induced increases in Cebpb mRNA expression (B) and C/EBPβ protein abundance (C) on day 7 after CCI or post-sham surgery in the ipsilateral L3/4 DRGs. Unilateral L3/4 DRGs from two mice were pooled together. n = 6 to 8 mice per group. One-way ANOVA followed by Tukey post hoc test, F4,16 = 17.041 in (B) and F4,19 = 9.614 in (C). **P < 0.01 compared to the vehicle (V) plus sham group. #P < 0.05 or ##P < 0.01 compared to the vehicle plus CCI group. (D to J) Effect of pre-microinjection of Cebpb siRNA, vehicle, or negative control siRNA into the ipsilateral L3/4 DRGs on paw withdrawal frequencies to mechanical stimuli (D and E), paw withdrawal latency to thermal stimulation (F), and paw jumping latency to cold stimulation (G) on the ipsilateral side and on basal paw withdrawal responses to mechanical (H and I) and thermal (J) stimuli on the contralateral side at different days after CCI or sham surgery. n = 10 mice per group. Two-way ANOVA followed by Tukey post hoc test, F4,199 = 37.159 in (D), F4,199 = 27.312 in (E), F4,199 = 37.336 in (F), F4,199 = 56.651 in (G), F4,199 = 2.071 in (H), F4,199 = 0.472 in (I), and F4,199 = 0.681 in (J). **P < 0.01 compared to the corresponding baseline (day –4). (K and L) Effect of pre-microinjection of Cebpb siRNA into the ipsilateral L3/4 DRGs on CCI-induced increases in the phosphorylation of ERK1/2 (p-ERK1/2) and abundance of GFAP in the ipsilateral L3/4 dorsal horn on day 5 post–spinal nerve ligation (SNL). Representative Western blots (K) and a summary of densitometric analysis (L) are shown. n = 6 to 8 mice per group. One-way ANOVA followed by Tukey post hoc test, F2,9 = 9.003 for p-ERK1, F2,9 = 8.899 for p-ERK2, F2,11 = 0.156 for ERK1, F2,11 = 0.192 for ERK2, and F2,11 = 8.163 for GFAP. *P < 0.05 compared to the corresponding vehicle plus sham group. #P < 0.05 compared to the corresponding vehicle plus CCI group.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Negative Control, Cell Culture, Western Blot, Microinjection, Expressing, Quantitative Proteomics, Phospho-proteomics, Ligation

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: Mean changes in locomotor function SEM given in parentheses. n = 10 mice per group; five trials.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Functional Assay, Negative Control, Saline

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A to G) Effect of DRG microinjection of Cebpb siRNA, vehicle, or negative control siRNA starting on day 5 after CCI on paw withdrawal frequencies to mechanical stimuli (A and B), paw withdrawal latency to thermal stimulation (C), and paw jumping latency to cold stimulation (D) on the ipsilateral side and on basal paw withdrawal responses to mechanical (E and F) and thermal (G) stimuli on the contralateral side on days 12 and 14 after CCI. n = 10 mice per group. Two-way ANOVA followed by Tukey post hoc test, F2,119 = 26.777 in (A), F2,119 = 31.439 in (B), F2,119 = 15.275 in (C), F2,119 = 18.089 in (D), F2,119 = 0.093 in (E), F2,119 < 0.001 in (F), and F2,119 = 0.157 in (G). **P < 0.01 compared to the vehicle plus CCI group at the corresponding time points.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Microinjection, Negative Control

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A and B) Cebpb mRNA expression (A) and C/EBPβ protein abundance (B) in the ipsilateral L3/4 DRGs 8 weeks after microinjection of AAV5-C/EBPβ or control AAV5-EGFP. n = 6 mice per group. Two-tailed, independent Student’s t test, *P < 0.05 compared to the AAV5-EGFP group. (C to F) Effect of microinjection of AAV5-C/EBPβ or AAV5-EGFP into the unilateral L3/4 DRGs on paw withdrawal frequencies to mechanical stimuli (C and D), paw withdrawal latency to thermal stimulation (E), and paw jumping latency to cold stimulation (F) on the ipsilateral and contralateral sides at the different weeks after microinjection. n = 10 mice per group. Two-way ANOVA followed by Tukey post hoc test, F3,239 = 42.76 in (C), F3,239 = 39.60 in (D), F3,239 = 59.08 in (E), and F1,119 = 49.879 in (F). **P < 0.01 compared to the control AAV5-EGFP group on the ipsilateral side at the corresponding time points. (G and H) Effect of microinjection of AAV5-C/EBPβ or AAV5-EGFP into the unilateral L3/4 DRGs on spontaneous ongoing pain as assessed by CPP paradigm. n = 16 mice per group. **P < 0.01 compared to the corresponding preconditioning (G) or the AAV5-EGFP group (H) by two-tailed, independent Student’s t test.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative Proteomics, Microinjection, Control, Two Tailed Test

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A and B) Ehmt2 mRNA expression (A), the abundance of G9a’s two protein isoforms (B), and the amount of H3K9me2-marked protein (B) in the ipsilateral L3/4 DRGs 7 days after CCI or sham surgery in the mice pre-microinjected with vehicle, negative control siRNA, or Cebpb siRNA into the ipsilateral L3/4 DRGs. Unilateral L3/4 DRGs from two mice were pooled together. n = 6 to 8 mice per group. One-way ANOVA followed by Tukey post hoc test, F4,16 = 33.704 for Ehmt2 mRNA, F4,19 = 16.879 for G9a-L, F4,19 = 28.072 for G9a-S, and F4,14 = 19.035 for H3K9me2. **P < 0.01 compared to the corresponding vehicle plus sham group; #P < 0.05 or ##P < 0.01 compared to the corresponding vehicle plus CCI group. L, long isoform; S, short isoform. (C and D) The amounts of Ehmt2 mRNA (C), G9a’s two protein isoforms (D), and H3K9me2 protein (D) in the injected L3/4 DRGs 8 weeks after microinjection of AAV5-C/EBPβ or control AAV5-EGFP. n = 6 to 8 mice per group. Two-tailed, independent Student’s t test, *P < 0.05 compared to the corresponding AAV5-EGFP group. (E) Coexpression of Cebpb mRNA with Ehmt2 mRNA in small, medium, and large DRG neurons. Gapdh mRNA was used a positive control. n = 3 biological replicates. M, ladder marker. (F) ChIP analysis of C/EBPβ binding to a fragment of the Ehmt2 gene distal promoter in the ipsilateral L3/4 DRGs on day 7 after CCI or sham surgery. Input, total purified fragments. Ipsilateral L3/4 DRGs from 3 mice were pooled together. n = 9 mice per group. (G to I) The abundance of C/EBPβ, G9a’s two protein isoforms, and H3K9me2 in mouse cultured DRG neurons transduced as indicated. Representative Western blots (G) and a summary of densitometric analysis [C/EBPβ (H) and G9a-L, G9a-S, and H3K9me2 (I)] are shown. n = 5 biological replicates per treatment. One-way ANOVA followed by Tukey post hoc test, F5,29 = 51.42 for C/EBPβ, F5,29 = 19.66 for G9a-L, F5,29 = 27.95 for G9a-S, and F5,29 = 10.16 for H3K9me2. **P < 0.01 compared to the corresponding naïve group; ##P < 0.01 compared to the corresponding C/EBPβ-treated group.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Negative Control, Injection, Microinjection, Control, Two Tailed Test, Positive Control, Marker, Binding Assay, Purification, Cell Culture, Western Blot

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A and B) Oprm1 and Kcan2 mRNA expression (A) and protein abundance (B) in the ipsilateral L3/4 DRGs 7 days after sham surgery or CCI in the mice pre-microinjected with vehicle, negative control siRNA, or Cebpb siRNA into the ipsilateral L3/4 DRGs. Unilateral L3/4 DRGs were pooled together. n = 6 to 8 mice per group. One-way ANOVA followed by Tukey post hoc test, F4,16 = 13.249 for Oprm1 mRNA, F4,16 = 14.464 for Kcna2 mRNA, F4,19 = 12.658 for MOR protein, and F4,19 = 11.820 for Kv1.2 protein. *P < 0.05 or **P < 0.01 compared to the corresponding vehicle plus sham group; #P < 0.05 compared to the corresponding vehicle plus CCI group. (C and D) Oprm1 and Kcna2 mRNA expression (C) and MOR and Kv1.2 protein abundance (D) in the injected L3/4 DRGs 8 weeks after microinjection of AAV5-C/EBPβ or control AAV5-EGFP. n = 6 to 8 mice per group. Two-tailed, independent Student’s t test, *P < 0.05 compared to the corresponding AAV5-EGFP group. (E and F) Coexpression of Cebpb mRNA with Oprm1 mRNA (E) or Kcna2 mRNA (F) in small, medium, and large DRG neurons. Gapdh mRNA was used as a positive control. n = 3 biological replicates per gene. (G and H) The abundance of C/EBPβ protein, G9a’s two protein isoforms, and H3K9me2 protein (G) and MOR and Kv1.2 protein (H) in cultured DRG neurons from adult wild-type (WT) (G9afl/fl mice) or G9a conditional knockout (KO) mice. The cultured neurons were collected 2 days after they were transduced with AAV5-EGFP or AAV5-C/EBPβ. n = 3 biological replicates per group. One-way ANOVA with Tukey post hoc test, F3,11 = 10.206 for C/EBPβ, F3,11 = 51.332 for G9a-L, F3,11 = 38.517 for G9a-S, F3,11 = 52.955 for H3K9me2, F3,11 = 27.503 for MOR, and F3,11 = 30.199 for Kv1.2. *P < 0.05 or **P < 0.01 compared to the corresponding AAV5-EGFP–treated WT mice; ##P < 0.01 compared to the corresponding the AAV5-C/EBPβ–treated WT mice. (I) Venn diagram of genes from microarray analysis of the DRG in the mice that overexpressed Ehmt2 (11) and from RNA sequencing data of the injured DRG in a neuropathic pain mouse model treated with a G9a inhibitor (21). About 254 genes overlapped between these two databases.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative Proteomics, Negative Control, Injection, Microinjection, Control, Two Tailed Test, Positive Control, Cell Culture, Knock-Out, Transduction, Microarray, RNA Sequencing

Journal: Science signaling

Article Title: The transcription factor C/EBPβ in the dorsal root ganglion contributes to peripheral nerve trauma–induced nociceptive hypersensitivity

doi: 10.1126/scisignal.aam5345

Figure Lengend Snippet: (A) Effect of pre-microinjection of Cebpb siRNA or vehicle into the ipsilateral DRG on morphine (1 mg/kg, subcutaneously) analgesia on the ipsilateral side 3 days after CCI. n = 9 mice per group. One-way ANOVA with Tukey post hoc test, F2,26 = 7.833 on the ipsilateral side and F2,26 = 0.969 on the contralateral side. *P < 0.05 compared to the corresponding vehicle plus sham group. #P < 0.01 compared to the corresponding vehicle plus CCI group. MPAE, maximal possible analgesic effect. (B) Effect of intraperitoneal injection with methylnaltrexone (2 mg/kg) on the Cebpb siRNA–produced antinociceptive effect on day 4 after CCI on the ipsilateral side of the Cebpb siRNA–treated group. Paw withdrawal latency to thermal stimulation was measured before and 30 min after drug administration on day 4 after CCI or sham surgery. n = 9 mice per group. Two-way ANOVA with Tukey post hoc test, F2,80 = 109.908 on the ipsilateral side and F2,80 = 0.718 on the contralateral side. **P < 0.01 compared to the corresponding vehicle plus sham group −1 day after CCI or sham surgery. #P < 0.05 compared to the vehicle plus CCI group before drug administration on day 4 after CCI or sham surgery. $P < 0.05 compared to the Cebpb siRNA plus CCI group before drug administration on day 4 after CCI or sham surgery.

Article Snippet: For siRNA injection, Cebpb siRNA (catalog no. sc-29862) and its negative control siRNA (catalog no. sc-44230) were purchased from Santa Cruz Biotechnology.

Techniques: Microinjection, Injection, Produced

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: SE-associated LINC01013 was transcriptionally activated by CEBPB in hPASMCs. A Prediction of candidate transcription factors and binding sites. (Transcription factor related databases: JASPAR: https://jaspar.elixir.no/ ; PROMO: https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ; GENECARD: https://www.genecards.org/ ; AnimalTFDB: http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/ ; Super enhancer related database: LncSEA: https://bio.liclab.net/LncSEA/ ). B The schematic diagram illustrates the LINC01013 promoter, divided into segments P1-P4, and the binding sites of transcription factors. C Four constituents (E1-E4) of SE region of LINC01013 derived from the WashU Epigenome Browser databases ( http://epigenomegateway.wustl.edu/browser/ ). D , E hPASMCs were subjected to ChIP analysis using antibodies against H3K27ac, H3K4me1 and CEBPB. The association with the SE region (D, E1-E4) and promoter region (E, P1-P4) of LINC01013 was quantified by RT‒qPCR ( n = 3). F hPASMCs were treated with CEBPB siRNA and subjected to ChIP analysis using antibodies against H3K27ac. The association with the E2 of SE (left) and P1-P3 promoter regions (right) of LINC01013 was quantified by RT-qPCR ( n = 3). G Schematic diagram of transcribing LINC01013 in hPASMCs. All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA or Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; IP, immunoprecipitation; IgG, Immunoglobulin G; TSS, transcription initiation site

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Binding Assay, Derivative Assay, Quantitative RT-PCR, Negative Control, Immunoprecipitation

Journal: The Journal of cell biology

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains.

doi: 10.1083/jcb.201807119

Figure Lengend Snippet: Figure 1. Amphiphysin drives membrane fission, while the N-BAR domain stabilizes membrane tubules. Mem- brane composition for vesicles in TEM: 80 mol% DOPC, 5 mol% PtdIns(4,5)P2, and 15 mol% DOPS. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 1 mol% Texas Red–DHPE. (A) Schematic of Amph-FL dimer. BAR domain: PDB 4ATM. SH3 domain: PDB 1BB9. (B–D) Negative stain TEM micrographs of 200 nm extruded vesicles before exposure to protein (B), after exposure to 26 µM N-BAR (C), and after exposure to 5 µM Amph-FL (D). Dashed boxes indicate zoomed regions to the right. Black arrows indi- cate membrane tubules; red arrowheads indicate fission vesi- cles. Yellow asterisks indicate small vesicles that are present in the vesicle population before protein exposure. (E) Histograms of vesicle diameters measured from electron micrographs. Ves- icles alone: n = 1,302 vesicles. 26 µM N-BAR: n = 462 vesicles. 5 µM Amph-FL: n = 1,071 vesicles. (F) Membrane release from SUPER templates, measured as Texas Red signal present in the supernatant after sedimentation of the SUPER templates. Membrane release in the absence of protein was measured and subtracted as background. Dots indicate data and lines indi- cate mean; n = 3 independent experiments. P value: one-tailed, unpaired Student’s t test. (B–D) Bars, 500 nm; insets, 200 nm. See also Fig. S1 and Videos 1, 2, 3, 4, and 5.

Article Snippet: The pCAG EN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1–256), tagged at the C terminus with mCherry, was a gift from T. Meyer, Stanford University, Stanford, CA (Addgene plasmid 85130).

Techniques: Membrane, Staining, Sedimentation, One-tailed Test

Journal: The Journal of cell biology

Article Title: BAR scaffolds drive membrane fission by crowding disordered domains.

doi: 10.1083/jcb.201807119

Figure Lengend Snippet: Figure 3. The disordered domain of amphiphysin alone drives membrane fission, but the N-BAR scaffold substantially enhances fission efficiency. Membrane composition in Amph CTD ΔSH3 tethered vesicle experiments: 76 mol% DOPC, 20 mol% DOGS- NTA-Ni, 2 mol% Oregon Green 488–DHPE, and 2 mol% DP-EG10-biotin. In tethered vesicle experiments with N-BAR-epsin CTD, DOGS-NTA-Ni was replaced with 5 mol% PtdIns(4,5)P2 and 15 mol% DOPS. SUPER template membrane composition: 79 mol% DOPC, 5 mol% PtdIns(4,5)P2, 15 mol% DOPS, and 1 mol% Texas Red–DHPE. (A) Schematic of Amph CTD ΔSH3. (B) Tethered vesicle fission experiments show that Amph CTD ΔSH3 forms highly curved fission products. (C) Summary of data from tethered vesicle fission exper- iments with Amph CTD ΔSH3 expressed as the ratio of the distribution area below 45 nm to the total distri- bution area (compare to Fig. 2 F). (D) Coverage of the membrane surface by Amph CTD ΔSH3 and Amph-FL as a function of protein concentration. Amph-FL data from Fig. 2 H. (E) Fraction of vesicle diameters below 45 nm generated by Amph CTD ΔSH3 and Amph-FL versus coverage of the membrane surface by proteins. Amph-FL fission data from Figs. 2 F and S2 M. Amph CTD ΔSH3 fission data from Fig. 3 C. (F) Schematic of N-BAR-epsin CTD chimera dimer. (G) Tethered vesicle fission measurements show that N-BAR-epsin CTD generates highly curved fission vesicle populations over the concentration range of 10–150 nM, similar to Amph-FL (compare to Fig. 2 D). (H) Summary of data from tethered vesicle fission experiments with N-BAR-epsin CTD, expressed as the ratio of the dis- tribution area below 45 nm to the total distribution area. Amph-FL and N-BAR data from Fig. 2 F. (I) SUP ER template membrane shedding experiments show that N-BAR-epsin CTD drives greater membrane release compared with N-BAR (compare to Fig. 1 F). Dots indicate data and lines indicate mean; n = 3 inde- pendent experiments. P value: one-tailed, unpaired Student’s t test. Amph CTD ΔSH3 markers in C and D and all markers in H represent mean ± first SD; n = 3 independent experiments. (J) Schematic of the N-BAR scaffold (EMDB 3192; Adam et al., 2015) with attachment points of some of the disordered domains marked (two per N-BAR dimer). Dashed circles indi- cate approximate volumes occupied by undeformed disordered domains. See also Figs. S2 and S3.

Article Snippet: The pCAG EN mammalian expression vector containing the N-BAR domain of human amphiphysin (residues 1–256), tagged at the C terminus with mCherry, was a gift from T. Meyer, Stanford University, Stanford, CA (Addgene plasmid 85130).

Techniques: Membrane, Protein Concentration, Generated, Concentration Assay, One-tailed Test