Journal: medRxiv

Article Title: Impairment of endothelial MerTK accelerates atherosclerosis development

doi: 10.1101/2025.04.14.25325845

Figure Lengend Snippet: ( A ) Body weight measurement for two months. ( B - C ) The visible formation of atherosclerotic plaque and H&E staining in aortic arch. ( D - G ) Immunostaining for the expression of IL-1β, MCP-1, ApoE, p22 phox and NF-κB in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model.

Article Snippet: The information of antibodies is shown as follows: IL-1β, p22 phox , p38 and α-SMA were from Cell Signaling; MCP-1 and TNF-α were from Invitrogen; ApoE was from Proteintech; p47 phox and gp91 phox were from SantaCruz Biotechnology; IFN-γ was from BiossUSA; and JNK was from Proteintech.

Techniques: Staining, Immunostaining, Expressing

Journal: medRxiv

Article Title: Impairment of endothelial MerTK accelerates atherosclerosis development

doi: 10.1101/2025.04.14.25325845

Figure Lengend Snippet: ( A - D ) Immunostaining for the expression of inflammation markers (TNF-α and IFN-γ), NADPH oxidase subunits (p47 phox and gp91 phox ), and MAPK family (ERK, p38 and JNK) in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model.

Article Snippet: The information of antibodies is shown as follows: IL-1β, p22 phox , p38 and α-SMA were from Cell Signaling; MCP-1 and TNF-α were from Invitrogen; ApoE was from Proteintech; p47 phox and gp91 phox were from SantaCruz Biotechnology; IFN-γ was from BiossUSA; and JNK was from Proteintech.

Techniques: Immunostaining, Expressing

Journal: bioRxiv

Article Title: Kinesin-1 mediates proper ER folding of the Ca V 1.2 channel and maintains mouse glucose homeostasis

doi: 10.1101/2024.06.24.600327

Figure Lengend Snippet: (A–C) Degradation assay in immunofluorescence against Ca V 1.2 (A) and Ca V 2.3 (B) of primary beta cells of the indicated genotypes after the CHX treatment of the indicated periods; accompanied by their quantification along with that of α-tubulin (C). Bars, 5 μm. * p < 0.05, *** p < 0.001, Welch’s t test at the indicated time; n = 6 (Ca V 1.2), 17–24 (Ca V 2.3), 5–6 (α-tubulin). (D and E) Brefeldin-A (BFA) washout assay with an LSM 5LIVE-Duo microscope, assessing the speeds of post-Golgi trafficking of Ca V 1.2-EGFP proteins expressed in primary beta cells of the indicated genotypes (D), accompanied by its quantification (E). Time after BFA washout is indicated. Bar, 5 μm. *p < 0.05, Welch’s t test, n = 11. Arrows, the timing of plasma membrane fusion. Corresponding to Movie EV4. (F) TIRF/STORM microscopy of a wild-type primary mouse beta cell immunolabeled against Ca V 1.2 and KIF5B. Scale bar, 5 μm. Arrows, colocalizing spots. (G) TIRF/STORM microscopy of primary mouse beta cells of the indicated genotypes immunolabeled against Ca V 1.2 (green) and Ca V 2.3 (magenta). Scale bars, 5 μm. (H) Schematic representation of STIP1-dependent Hsp70-to-Hsp90 chaperone exchange machinery. (I and J) z -projection of proximity ligation assay in CT and cKO primary beta cells showing the protein binding between Ca V 1.2 and the indicated Hsp proteins (I); accompanied by quantification (J). ** p < 0.01; *** p < 0.001; Welch’s t test, n = 6. (K) Immunoblotting of scramble control (SC) and STIP1-knockdown (KD) MIN6 cells against the indicated epitopes. Note that STIP1 deficiency induced downregulation of Ca V 1.2 and BK Ca proteins. Reproduced twice.

Article Snippet: A rabbit anti-Ca V 1.2 antibody (N-17-R, #sc-16229-R, RRID:AB_2228387), a rabbit anti-K ir 6.2 antibody (H-55; #sc-20809; RRID:AB_2130466), a goat anti-PIP5Kα (PIPKIα) antibody (M-20, #sc-11775; RRID:AB_2268303), and a goat anti-SUR1 antibody (N-18, #sc-11226; RRID:AB_2130475) were purchased from Santa Cruz Biotechnology; a mouse anti-PIP 2 IgM antibody (#Z-A045, RRID:AB_427211) was from Echelon Research labs; a rabbit anti-GFP antibody (#598, RRID:AB_591819) was from MBL; a mouse anti-LC3 antibody (Clone LC3-1703, #CTB-LC3-2-IC, RRID:AB_10707197) was from Cosmo Bio; a rabbit anti-BK Ca (K Ca 1.1) antibody (#APC-151, RRID:AB_10915895) and a rabbit anti-Ca V 2.3 antibody (#ACC-006, RRID:AB_2039777) were from Alomone Labs; a mouse anti-syntaxin-1 antibody (#MAB336, RRID:AB_2196527) was from Millipore; a mouse anti-Na/K ATPase beta 2 antibody (#610914; RRID:AB_398231) and a mouse anti-paxillin antibody (#610051, RRID:AB_397463) were from BD Transduction Labs; a mouse anti-Hsc70/Hsp70 antibody (Clone BB70, #ADI-SPA-822-D, RRID:AB_2039252) and a rat anti-Hsp90 antibody (Clone 16F1, #ADI-SPA-835-D, RRID:AB_2039281) were from Enzo; a rabbit anti-STIP1 antibody (#15218-1-AP; RRID:AB_2255518) and a rabbit anti-calnexin-1 (CANX) antibody (#10427-2-AP, RRID:AB_2069033) were from Proteintech; a mouse anti-derlin-1 antibody (#SAB4200148, RRID:AB_10624068), an anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and an anti-insulin antibody (Clone K36aC10, #I-2018, Sigma Aldrich; RRID:AB_260137) were from Sigma Aldrich; a rabbit anti-phospho Src antibody for pSFK (#ab32078; RRID:AB_2286707) was from Epitomics/Abcam; and a rabbit anti-KIF5B antibody (RRID:AB_2571745) was previously described ( Tanaka et al ., 1998 ).

Techniques: Degradation Assay, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Immunolabeling, Proximity Ligation Assay, Protein Binding, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Kinesin-1 mediates proper ER folding of the Ca V 1.2 channel and maintains mouse glucose homeostasis

doi: 10.1101/2024.06.24.600327

Figure Lengend Snippet: (A and B) Rescue of Ca V 1.2 degradation in cKO primary beta cells in the presence of CHX by leupeptin (Leu) or MG-132 (MG) for 4 h. ns, p > 0.05; * p < 0.05; one-way ANOVA, n = 12. (C and D) Vesicle IP of MG-132-treated MIN6 cell lysates transduced with scrambled control (SC) and KIF5B-knockdown (KD) miRNAs, precipitated using Ca V 1.2 or K ir 6.2 antibodies or normal rabbit IgG (NRG) and immunoblotted for the indicated proteins (C), accompanied by quantification of Ca V 1.2-coprecipitated fractions (D). Note that the Ca V 1.2-binding capacities of derlin-1, calnexin-1, and Hsp90 chaperones and that of the adaptor protein STIP1 in KD cell lysates were significantly lower than those in SC cell lysates. (E) Vesicle IP of the MG-132-treated MIN6 cell lysates among the KIF5B KD system against STIP1. Note that the Hsp90 level in the STIP1 immunoprecipitants (IP) was greatly decreased by KIF5B deficiency. Repeated twice. (F) Schematic representation of the working hypothesis on differential KIF5B- and heat-shock-protein (Hsp)-dependencies of opposing ER clients Ca V 1.2 and K ir 6.2 in control (CT) and KIF5B conditional knockout (cKO) mouse beta cells. In cKO cells, Ca V 1.2 fails in chaperone exchange to undergo ERAD-mediated degradation, but K ir 6.2 is intact because it is independent on the KIF5B–Hsp machinery. (G and H) Ca V 1.2 immunocytochemistry of MIN6 cells that had been transduced with EYFP-KIF5B and/or TagRFP-Hsc70 or without them (NT; G); accompanied by their quantification (H). Scale bar, 5 μm. ns, p > 0.05; ** p < 0.01, one-way ANOVA, n = 5–13. Arrow in G, enhanced Ca V 1.2 expression according to dual overexpression. (I) Vesicle IP of non-transduced (NT) and KIF5B- and Hsc70-overexpressing (K5+H70 OE) MIN6 cell lysates against Ca V 1.2. Asterisks, tagged protein bands. The tagRFP-Hsc70 band was overlapped with a band of possibly ubiquitinated form. Reproduced twice.

Article Snippet: A rabbit anti-Ca V 1.2 antibody (N-17-R, #sc-16229-R, RRID:AB_2228387), a rabbit anti-K ir 6.2 antibody (H-55; #sc-20809; RRID:AB_2130466), a goat anti-PIP5Kα (PIPKIα) antibody (M-20, #sc-11775; RRID:AB_2268303), and a goat anti-SUR1 antibody (N-18, #sc-11226; RRID:AB_2130475) were purchased from Santa Cruz Biotechnology; a mouse anti-PIP 2 IgM antibody (#Z-A045, RRID:AB_427211) was from Echelon Research labs; a rabbit anti-GFP antibody (#598, RRID:AB_591819) was from MBL; a mouse anti-LC3 antibody (Clone LC3-1703, #CTB-LC3-2-IC, RRID:AB_10707197) was from Cosmo Bio; a rabbit anti-BK Ca (K Ca 1.1) antibody (#APC-151, RRID:AB_10915895) and a rabbit anti-Ca V 2.3 antibody (#ACC-006, RRID:AB_2039777) were from Alomone Labs; a mouse anti-syntaxin-1 antibody (#MAB336, RRID:AB_2196527) was from Millipore; a mouse anti-Na/K ATPase beta 2 antibody (#610914; RRID:AB_398231) and a mouse anti-paxillin antibody (#610051, RRID:AB_397463) were from BD Transduction Labs; a mouse anti-Hsc70/Hsp70 antibody (Clone BB70, #ADI-SPA-822-D, RRID:AB_2039252) and a rat anti-Hsp90 antibody (Clone 16F1, #ADI-SPA-835-D, RRID:AB_2039281) were from Enzo; a rabbit anti-STIP1 antibody (#15218-1-AP; RRID:AB_2255518) and a rabbit anti-calnexin-1 (CANX) antibody (#10427-2-AP, RRID:AB_2069033) were from Proteintech; a mouse anti-derlin-1 antibody (#SAB4200148, RRID:AB_10624068), an anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and an anti-insulin antibody (Clone K36aC10, #I-2018, Sigma Aldrich; RRID:AB_260137) were from Sigma Aldrich; a rabbit anti-phospho Src antibody for pSFK (#ab32078; RRID:AB_2286707) was from Epitomics/Abcam; and a rabbit anti-KIF5B antibody (RRID:AB_2571745) was previously described ( Tanaka et al ., 1998 ).

Techniques: Transduction, Control, Knockdown, Binding Assay, Knock-Out, Immunocytochemistry, Expressing, Over Expression

Journal: Nature chemical biology

Article Title: Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools.

doi: 10.1038/s41589-023-01381-8

Figure Lengend Snippet: Fig. 3 | Plasma membrane and the Golgi pools of β1AR function differently in adult mouse cardiomyocytes. a, Representative western blots of phosphorylation profiles of RyR2, TnI and PLB regulated by β1AR in adult cardiomyocytes derived from WT mice, treated with 10 μM corticosterone (Cortico). Adult cardiomyocytes were pretreated with 10 μM β2AR-selective antagonist ICI-118551 (ICI) to isolate the function of β1ARs. Membrane-permeable β1AR-selective agonist, dobutamine (Dob), promotes PLB phosphorylation independent of OCT3. b–d, Quantification of immunoblots of pRyR2 Ser2808, p-TnI Ser23/Ser24 and pPLB Ser16/Thr17 normalized to the protein level of CSQ2 and then reported as a percentage of the highest value in the groups.

Article Snippet: Rabbit anti-calsequestrin 2 (CSQ2) antibody (18422-1-AP) and rabbit anti-SLC22A1 antibody (24617-1-AP) are from Proteintech.

Techniques: Clinical Proteomics, Membrane, Western Blot, Phospho-proteomics, Derivative Assay