Journal: Biochemical and biophysical research communications

Article Title: Mitochondrial calcium homeostasis mediated by estradiol contributes to atrial fibrillation protection.

doi: 10.1016/j.bbrc.2025.152050

Figure Lengend Snippet: Fig. 1. OVX increases atrial fibrillation susceptibility and disrupts mitochondrial calcium homeostasis. (A) Representative ECG recordings from the control, AF, and AF + OVX groups, showing increased AF susceptibility in the OVX group. (B) Quantification of E2 concentration in different groups. (C) Quantification of Ca2+ content among different groups, showing a significant decrease in calcium load in the AF + OVX group. (D, E) Detection of mitochondrial membrane potential changes between groups using JC-1 monomer probe.Data are presented as mean ± SEM. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.(F–H) Representative of immunofluorescence staining for MICU1, NCX, and LETM1.

Article Snippet: The antibody was diluted in PBS: LETM1 (16024-1-AP, Proteintech, 1/200), MICU1 (DF14914, Affinity, 1/100), NCX (bs-1550R, Bioss, 1/200), and incubated at 4 ◦C overnight.

Techniques: Control, Concentration Assay, Membrane, Immunofluorescence, Staining

Journal: Cell chemical biology

Article Title: Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

doi: 10.1016/j.chembiol.2025.04.004

Figure Lengend Snippet: Figure 5. LETM1 as USP30 interactor (A and B) Proximity ligation assay (PLA) in HEK293 cells. HEK293 cells transiently transfected with FLAG-USP30, with PLA reaction between anti-FLAG antibody and anti-LETM1 antibody (A) or an anti-TOMM20 antibody (B). Yellow punctate signals represent the reported interactions, and blue signals indicate nuclei. A single antibody control (none) was included. Scale bar: 50 μm. (C) Confocal images of HEK293 cells +/− CCCP transiently expressing FLAG-USP30 and endogenous LETM1 protein. Anti-FLAG antibody (red), anti-LETM1 antibody (green), and DAPI (blue) for nuclei visualization. Scale bars: 10 μm. (D and E) HEK293T cells transiently expressing FLAG-USP30 (D) USP30 was pulled down using an anti-FLAG antibody. (E) LETM1 was pulled down using an anti- LETM1 antibody. (F) SH-SY5Y cell LETM1 pull-down using anti-LETM1 antibody.

Article Snippet: LETM1 Proteintech CAT# 16024-1-AP Tomm20 (D8T4N) Cell signalling technology CAT# 42406 Flag Sigma Aldrich CAT# F3040 β-actin (8H10D10) Cell signalling technology CAT# 3700 Anti-HA (12CA5) Roche CAT# 11666606001 Ubiquitin (FK1) Sigma-Aldrich CAT# 04-262 FKBP8 Proteintech CAT# 11173-1-AP S65 Phospho-ubiquitin Cell signalling technology CAT# 62802S IgG Control (Rabbit-DA1E) Cell signalling technology CAT# 3900S IRDye® 800CW Streptavidin LICORbio CAT# 926-32230 IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody LICORbio CAT# 926-32210 IRDye® 800CW Donkey anti-Goat IgG Secondary Antibody LICORbio CAT# 926-32214 IRDye® 680CW Goat anti-Mouse IgG Secondary Antibody LICORbio CAT# 926-68070 IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody LICORbio CAT# 926-32211 IRDye® 680CW Goat anti-Rabbit IgG Secondary Antibody LICORbio CAT# 926-68071 Chemicals, peptides, and recombinant proteins DME Medium - high glucose Sigma Aldrich CAT# D6546 Fetal Bovine Serum (FBS) Gibco CAT# 10100147 GlutaMAX Supplement Gibco CAT# 35050061 Poly-D-Lysine Gibco CAT# A3890401 PhosSTOP phosphatase inhibitor Roche CAT# 4906837001 cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Roche CAT# 11836170001 PierceTM DTT (Dithiothreitol) ThermoFisher Scientific CAT# 20290 Iodoacetamide Sigma-Aldrich CAT# A3221 Hydrogen peroxide (H2O2), 30% (wt/wt) Sigma-Aldrich CAT# H1009-100ML Trifluoroacetic acid Sigma-Aldrich CAT# 91707 Formic acid Sigma-Aldrich CAT# 56302 Biotin-phenol Sigma-Aldrich CAT# SML2135 Sodium ascorbate Sigma-Aldrich CAT# A7631 Trolox Sigma-Aldrich CAT# 238813 Sodium azide Sigma-Aldrich CAT# S2002 Phosphoric acid Sigma-Aldrich CAT# 49685 SDS, 20% Solution, RNase-free ThermoFisher Scientific CAT# AM9820 RIPA Lysis and Extraction Buffer Thermo Scientific CAT# 89901 InstantBlue Coomassie Protein Stain (ISB1L) Abcam CAT# ab119211 L-Glutamine (200 mM) Gibco CAT# 25030149 Eagle’s Minimum Essential Medium Sigma CAT# M2279 (Continued on next page) ll OPEN ACCESSResource Cell Chemical Biology 32, 1–16.e1–e8, May 15, 2025 e1

Techniques: Proximity Ligation Assay, Transfection, Control, Expressing

Journal: Cell chemical biology

Article Title: Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

doi: 10.1016/j.chembiol.2025.04.004

Figure Lengend Snippet: Figure 6. LETM1 and FKBP8 deubiquitination are USP30 dependent (A) HA-ubiquitin pull-down using anti-HA antibody. HEK293T cells transiently expressing HA-Ub treated with 10 μM CCCP +/− compound 39. (B) HA-ubiquitin pull-down using anti-HA antibody. HEK293T cells transiently transfected with scrambled or USP30 siRNA and treated with 10 μM CCCP. (C and D) Tandem ubiquitin binding entity (TUBE) pull-down in HEK293T cells treated with 10 μM CCCP +/− compound 39.

Article Snippet: LETM1 Proteintech CAT# 16024-1-AP Tomm20 (D8T4N) Cell signalling technology CAT# 42406 Flag Sigma Aldrich CAT# F3040 β-actin (8H10D10) Cell signalling technology CAT# 3700 Anti-HA (12CA5) Roche CAT# 11666606001 Ubiquitin (FK1) Sigma-Aldrich CAT# 04-262 FKBP8 Proteintech CAT# 11173-1-AP S65 Phospho-ubiquitin Cell signalling technology CAT# 62802S IgG Control (Rabbit-DA1E) Cell signalling technology CAT# 3900S IRDye® 800CW Streptavidin LICORbio CAT# 926-32230 IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody LICORbio CAT# 926-32210 IRDye® 800CW Donkey anti-Goat IgG Secondary Antibody LICORbio CAT# 926-32214 IRDye® 680CW Goat anti-Mouse IgG Secondary Antibody LICORbio CAT# 926-68070 IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody LICORbio CAT# 926-32211 IRDye® 680CW Goat anti-Rabbit IgG Secondary Antibody LICORbio CAT# 926-68071 Chemicals, peptides, and recombinant proteins DME Medium - high glucose Sigma Aldrich CAT# D6546 Fetal Bovine Serum (FBS) Gibco CAT# 10100147 GlutaMAX Supplement Gibco CAT# 35050061 Poly-D-Lysine Gibco CAT# A3890401 PhosSTOP phosphatase inhibitor Roche CAT# 4906837001 cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail Roche CAT# 11836170001 PierceTM DTT (Dithiothreitol) ThermoFisher Scientific CAT# 20290 Iodoacetamide Sigma-Aldrich CAT# A3221 Hydrogen peroxide (H2O2), 30% (wt/wt) Sigma-Aldrich CAT# H1009-100ML Trifluoroacetic acid Sigma-Aldrich CAT# 91707 Formic acid Sigma-Aldrich CAT# 56302 Biotin-phenol Sigma-Aldrich CAT# SML2135 Sodium ascorbate Sigma-Aldrich CAT# A7631 Trolox Sigma-Aldrich CAT# 238813 Sodium azide Sigma-Aldrich CAT# S2002 Phosphoric acid Sigma-Aldrich CAT# 49685 SDS, 20% Solution, RNase-free ThermoFisher Scientific CAT# AM9820 RIPA Lysis and Extraction Buffer Thermo Scientific CAT# 89901 InstantBlue Coomassie Protein Stain (ISB1L) Abcam CAT# ab119211 L-Glutamine (200 mM) Gibco CAT# 25030149 Eagle’s Minimum Essential Medium Sigma CAT# M2279 (Continued on next page) ll OPEN ACCESSResource Cell Chemical Biology 32, 1–16.e1–e8, May 15, 2025 e1

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Binding Assay

Journal: Cell chemical biology

Article Title: Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

doi: 10.1016/j.chembiol.2025.04.004

Figure Lengend Snippet: Figure 5. LETM1 as USP30 interactor (A and B) Proximity ligation assay (PLA) in HEK293 cells. HEK293 cells transiently transfected with FLAG-USP30, with PLA reaction between anti-FLAG antibody and anti-LETM1 antibody (A) or an anti-TOMM20 antibody (B). Yellow punctate signals represent the reported interactions, and blue signals indicate nuclei. A single antibody control (none) was included. Scale bar: 50 μm. (C) Confocal images of HEK293 cells +/− CCCP transiently expressing FLAG-USP30 and endogenous LETM1 protein. Anti-FLAG antibody (red), anti-LETM1 antibody (green), and DAPI (blue) for nuclei visualization. Scale bars: 10 μm. (D and E) HEK293T cells transiently expressing FLAG-USP30 (D) USP30 was pulled down using an anti-FLAG antibody. (E) LETM1 was pulled down using an anti- LETM1 antibody. (F) SH-SY5Y cell LETM1 pull-down using anti-LETM1 antibody.

Article Snippet: Depending on the sample, the cells were then incubated with the following primary antibodies: ANTI-FLAG® M1 (1:1000) (Sigma Aldrich F3040), LETM1 (1:100) (Proteintech, 16024-1-AP), and TOMM20 (1:100) (Cell Signaling, 42406) at 4◦C overnight.

Techniques: Proximity Ligation Assay, Transfection, Control, Expressing

Journal: Cell chemical biology

Article Title: Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

doi: 10.1016/j.chembiol.2025.04.004

Figure Lengend Snippet: Figure 6. LETM1 and FKBP8 deubiquitination are USP30 dependent (A) HA-ubiquitin pull-down using anti-HA antibody. HEK293T cells transiently expressing HA-Ub treated with 10 μM CCCP +/− compound 39. (B) HA-ubiquitin pull-down using anti-HA antibody. HEK293T cells transiently transfected with scrambled or USP30 siRNA and treated with 10 μM CCCP. (C and D) Tandem ubiquitin binding entity (TUBE) pull-down in HEK293T cells treated with 10 μM CCCP +/− compound 39.

Article Snippet: Depending on the sample, the cells were then incubated with the following primary antibodies: ANTI-FLAG® M1 (1:1000) (Sigma Aldrich F3040), LETM1 (1:100) (Proteintech, 16024-1-AP), and TOMM20 (1:100) (Cell Signaling, 42406) at 4◦C overnight.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Binding Assay