Journal: Anais da Academia Brasileira de Ciencias

Article Title: Proteolytic characterization of a novel enzymatic extract from Bromelia serra leaves.

doi: 10.1590/0001-3765202220201871

Figure Lengend Snippet: Figure 1. SDS-polyacrylamide gel electrophoresis (PAGE). Lane 1: standard protein markers; lane 2: crude ex tract; lane 3: enzymatic extract; lane 4: enzymatic extract with 2-mercaptoethanol (a). Zymography. Lane 1: enzymatic extract; Lane 2: standard protein markers (b). IEF. Lane 1, enzymatic extract of BS; lane 2, pI markers: amyloglucosidase (pI 3.50), trypsin inhibitor (p I 4.55), b-lactoglobulin A (pI 5.20), carbonic anhydrase II (pI 5.85), carbonic anhydrase I (pI 6.55), myoglobin (pI 6.85 and 7.35), lectins from Lens culinaris (pI 8.15, 8.45 and 8.65), and trypsinogen (pI 9.30) (c).

Article Snippet: Isoelectric focusing (IEF) was developed on 5% polyacrylamide gels containing broad pH range ampholytes (Biolyte 3–10, Bio-Rad) in a Mini IEF Cell (Model 111, Bio-Rad).

Techniques: Polyacrylamide Gel Electrophoresis, Zymography

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

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 for the indicated periods; accompanied by their quantification along with that of α-tubulin ( C ). Bars, 5 μm. * P = 0.02561, *** P = 0.00292; one-sided unpaired Welch’s t test at the indicated time points; n = 6 cells (Ca V 1.2), 17–24 cells (Ca V 2.3), 5–7 cells (α-tubulin). Data are represented by the mean ± SEM. ( D , 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 = 0.0207, one-sided unpaired Welch’s t test, n = 11 cells. Arrows, the timing of plasma membrane fusion. Data are represented by the mean ± SEM. Corresponding to Movie . ( 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 , J ) Proximity ligation assay in CT and cKO primary beta cells with z- projection views, indicating protein binding signals between Ca V 1.2 and the indicated Hsp proteins ( I ); accompanied by quantification ( J ). Scale bar, 5 μm. ** P = 0.0122; *** P = 8.42 × 10 −5 ; one-sided unpaired Welch’s t test, n = 6 cells. Data are represented by the mean ± SEM. ( 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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

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

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

Figure Lengend Snippet: ( A , 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. Scale bar, 5 μm. ns, P = 0.4331; * P = 0.0112; one-way ANOVA with Dunnett’s multiple comparison test, n = 12 cells. Data are represented by the mean ± SEM. ( C , 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 ). ns 1) , P = 0.0634; ns 2) , P = 0.2434; * P = 0.0235; ** 1) P = 0.0014; ** 2) P = 0.0016; ** 1) P = 0.0092; *** P = 3.54 × 10 −4 ; one-sided unpaired Welch’s t test between KD and SC, n = 3–4 biological replicates. Data are represented by the mean ± SEM. 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 , 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 1) , P = 0.4266; ns 2) , P > 0.9999; ** P = 0.0017; one-way ANOVA with Dunnett’s multiple comparison between KD and SC, n = 5–13 biological replicates. Data are represented by the mean ± SEM. 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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

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

Journal: EMBO Reports

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

doi: 10.1038/s44319-024-00246-y

Figure Lengend Snippet: Reagents and tools table

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), a mouse anti-α-tubulin antibody (Clone DM1A; #CP06; RRID:AB_2617116), and a mouse 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, ).

Techniques: Knock-Out, Recombinant, Plasmid Preparation, Knockdown, Expressing, Transduction, Sequencing, Software, Enzyme-linked Immunosorbent Assay, Blocking Assay, Transfection, Isolation, Western Blot