jip4 antibody  (Santa Cruz Biotechnology)

Journal: Cell communication and signaling : CCS

Article Title: The SIRT5-JIP4 interaction promotes osteoclastogenesis by modulating RANKL-induced signaling transduction.

doi: 10.1186/s12964-024-02021-x

Figure Lengend Snippet: Fig. 7 Identification of JIP4 as a target protein of SIRT5. A-B Protein samples from BMDMs treated with RANKL and M-CSF for 2 days were collected, and SIRT5 was immunoprecipitated (IP-SIRT5) for subsequent Western blot verification (A). The gel strip of the IP-SIRT5 sample was subjected to Coomassie Brilliant Blue staining, and the band between 25 kD and 35 kD, indicated by black boxes, represented the band of SIRT5 (B). The entire gel strip was further subjected to protein mass spectrometry analysis. C After excluding non-specifically bound proteins detected in the IgG negative control, subcellular localization of proteins detected in the IP-SIRT5 mass spectrometry was statistically analyzed and presented using a Venn diagram. The results show the quantity of proteins localized in the cytoplasm, mitochondria, nucleus, and other/unknown locations. D Representative immunofluorescence image of SIRT5 (green), the mitochondrial marker TOMM20 (red) and DAPI (blue) in BMDMs treated with MCSF or MCSF and RANKL for 1 day (scale bars, 20 μm/ 2 μm). E-F The line charts represent the fluorescence intensity of SIRT5 and TOMM20, presenting the distance from α to γ in BMDMs treated with MCSF (E) or MCSF and RANKL (F). G The top five cytoplasmic proteins ranked by protein scores in the IP-MS results were listed. H BMDMs were transfected with Sirt5-overexpressing lentivirus, treated with MCSF and RANKL for 2 days, and the protein levels of JIP4 and SIRT5 in the IP-SIRT5 products were detected

Article Snippet: Lysates were incubated with either IgG, SIRT5, or JIP4 antibody for 2 h and then with protein A/G-agarose beads (Santa Cruz, Dallas, USA) overnight at 4 °C.

Techniques: Immunoprecipitation, Western Blot, Stripping Membranes, Staining, Mass Spectrometry, Negative Control, Immunofluorescence, Marker, Fluorescence, Protein-Protein interactions, Transfection

Journal: Cell communication and signaling : CCS

Article Title: The SIRT5-JIP4 interaction promotes osteoclastogenesis by modulating RANKL-induced signaling transduction.

doi: 10.1186/s12964-024-02021-x

Figure Lengend Snippet: Fig. 8 SIRT5 promotes the phosphorylation of p38 and JNK through its interaction with JIP4. A Relative mRNA expression of JIP1, JIP2, JIP3, JIP4 in BMDMs after 2-day treatment of MCSF and RANKL (n = 3). B Relative mRNA expression of JIP4 and osteoclastogenesis-related genes including early osteoclastic differentiation markers (Nfatc1 and PU.1), osteoclast fusion markers (Dc-stamp and Oc-stamp), and osteoclast functional markers (Trap and Ctsk) (n = 3). C Expression levels of JIP4 and osteoclastogenesis-related proteins in CON, sh-JIP4 BMDMs after 4 days RANKL induction. D-E CON, sh-JIP4 BMDMs were induced with MCSF and RANKL for 5 days, and then TRAP staining was used to identify TRAP-positive multinucleated cells (D). Scale bar, 200 μm. The number of TRAP-positive multinucleated cells was calculated and presented graphically (n = 6) (E). F-G Images showing bone resorption areas in the CON and sh-JIP4 groups (F). Scale bar, 200 μm. The resorption pit areas were quantified. (n = 4) (G). H After exposing CON and sh-JIP4 BMDMs to RANKL for 0, 5 and 10 min, the phosphorylation levels of p38 and JNK were detected by Western blot. I After treating CON and sh-Sirt5 BMDMs with MCSF and RANKL for 2 days, protein levels of JIP4, p38, and SIRT5 in the IP-JIP4 products of BMDMs were examined. J After exposing CON and sh-Sirt5 BMDMs to RANKL for 0, 5 and 10 min, the phosphorylation levels of p38 and JNK were detected by Western blot. The data are presented as means ± SD. *p < 0.05, **p < 0.01 vs. CON, ***p < 0.001 vs. CON + RANKL

Article Snippet: Lysates were incubated with either IgG, SIRT5, or JIP4 antibody for 2 h and then with protein A/G-agarose beads (Santa Cruz, Dallas, USA) overnight at 4 °C.

Techniques: Phospho-proteomics, Expressing, Functional Assay, Staining, Western Blot

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 1. JIP3 and JIP4 induce dynein activity in vitro and in cells. (A) Interaction sites for motor proteins mapped within the N-terminal regions of JIP3 and JIP4. KIF5, kinesin-1 heavy chain; DLIC, dynein light intermediate chain; DHC, dynein heavy chain; p150, dynactin p150Glued subunit; KLC, kinesin light chain. (B)

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Activity Assay, In Vitro

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 2. JIP3 and JIP4 interact with dynein on autolysosomes. (A) Quantification of PLA (detects two proteins within 40 nm of one another) between endogenous dynein (DIC) and endogenous JIP3 or JIP4. Negative control (Neg.) is missing one primary antibody. n = 20–21 neurons; one-way ANOVA with Tukey’s multiple comparisons test (JIP3 versus JIP4, P = 0.6763; JIP3 versus Neg., P < 0.0001; JIP4 versus Neg., P < 0.0001). (B) Schematic illustrating the formation of autolysosomes via fusion between an LC3+ AV and a LAMP1+ lysosome. (C) Schematic illustrating our PLA assay—probing for close apposition between endogenous DIC and endogenous JIP3 or JIP4—alongside colocalization with exogenously expressed HT-LC3 and endogenous LAMP1. (D–F) Example micrographs and quantifications showing colocalization between LC3, LAMP1, and JIP3-DIC or JIP4-DIC PLA puncta. n = 20 neurons; one-way ANOVA with Tukey’s multiple comparisons test; JIP3 (LC3 versus LC3 + LAMP1, P < 0.0001; LC3 versus LAMP1, P = 0.7236; LAMP1 versus LC3 + LAMP1, P < 0.0001); JIP4 (LC3 versus LC3 + LAMP1, P < 0.0001; LC3 versus LAMP1, P = 0.9879; LAMP1 versus LC3 + LAMP1, P < 0.0001). (G and H) Time series demonstrating JIP3 and JIP4 comigration with LC3 and LAMP1 in axons. Bars represent mean ± SEM. ns, not significant; ****, P < 0.0001.

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Negative Control

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 3. ARF6 regulates AV and lysosome retrograde transport. (A) Schematic of the middle regions of JIP3 and JIP4 demonstrating interactions with ARF6. (B) Schematic illustrating the effects of GTP-locked ARF6Q97L and GDP-locked ARF6QL on JIP3/4 binding interactions as described by Montagnac et al. (2009). (C) Representative Western blot and quantification of ARF6 knockdown in rat neural cells (PC12). n = 8. Samples were normalized using Revert 700 Total Protein Stain (LI-COR). (D and E) Fraction of LC3 or LAMP1 puncta moving ≥10 µm net displacement over a 2-min video (either direction). Mock, cells treated alongside others but without any added siRNA. siRNA, cells treated with ARF6 siRNA. Rescue, cells treated with ARF6 siRNA and simultaneously transfected with siRNA-resistant CFP-ARF6WT. n = 15–16 neurons; one-way ANOVA with Tukey’s multiple comparisons test. LC3: Mock versus siRNA, P = 0.0132; Mock versus Rescue, P = 0.3770; siRNA versus Rescue, P = 0.0002. LAMP1: Mock versus siRNA, P = 0.0162; Mock versus Rescue, P = 0.3677; siRNA versus Rescue, P = 0.0003. (F and G) Fraction of retrograde runs (≥10 µm net displacement towards the soma) over all events (including those that moved <10 µm) for LC3 and LAMP1 puncta. n = 15–16 neurons; one-way ANOVA with Holm–Sidak’s multiple comparisons test. LC3: Mock versus siRNA, P = 0.0018; Mock versus Rescue, P = 0.5109; siRNA versus Rescue, P = 0.0003. LAMP1: Mock versus siRNA, P = 0.0050; Mock versus Rescue, P = 0.4563; siRNA versus Rescue, P = 0.0008. (H and I) Fraction of anterograde runs (≥10 µm net displacement toward the axon tip) over all events (including those that moved

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Binding Assay, Western Blot, Knockdown, Staining, Transfection

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 5. ARF6 induces the recruitment of JIP3/4 to MTs. (A) Example kymographs showing the activity of JIP3- and JIP4-containing complexes in the presence of CFP-ARF6QL. (B and C) Quantification of the number of total landing events (includes runs and non-motile binding events) for JIP3- and JIP4-

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Activity Assay, Binding Assay

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 6. RAB10 knockdown disrupts AV and lysosome motility. (A) Middle region of JIP3 and JIP4 illustrating putative interaction with RAB10. (B and C) Fraction of autophagosomes (HT-LC3 only), autolysosomes (LC3 + LAMP1), or lysosomes (endogenous LAMP1 only) in fixed cells colocalized with EGFP-RAB10.

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Knockdown

Journal: The Journal of cell biology

Article Title: Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

doi: 10.1083/jcb.202301084

Figure Lengend Snippet: Figure 7. Integrated model of autophagosome, autolysosome, and lysosome transport along axons. (A) GTP-bound ARF6 is enriched on AV membranes through local GEF activity, where it can recruit interaction partners JIP3 or JIP4. JIP3/4 in turn recruits dynactin and dynein, leading to activation of the minus- end-directed retrograde motility of autolysosomes. RAB10 may induce anterograde transit, possibly favoring complex formation between JIP3 or JIP4 with JIP1 and kinesin-1. Additional motor complexes have also been implicated in AV or lysosome transport; we highlight a few complementary complexes on the left (Pu et al., 2015; Keren-Kaplan et al., 2022; Cason et al., 2021; Willett et al., 2017; Kumar et al., 2022; Wong and Holzbaur, 2014; Fu et al., 2014; Jongsma et al., 2020; Rosa-Ferreira et al., 2018; Far´ıas et al., 2017; Guardia et al., 2016; Keren-Kaplan and Bonifacino, 2021). (B) This pathway may be disrupted via hyper- phosphorylation of RABs. Disease-causing mutations in LRRK2 kinase result in increased phospho-RABs and also increased recruitment of kinesin-1 to the AV membrane (Boecker et al., 2021). The resulting loss of AV motility can be rescued experimentally by expression of GTP-locked ARF6 (Dou et al., 2023); thus these motor-regulatory mechanisms are interconnected and possibly competitive.

Article Snippet: Neurons were transfected (Lipofectamine 2000) with 0.3 μg EGFP plasmid (for GFP fill) and 0.35 μg HT LC3 following the above protocol, and then 24 h later (DIV 7–8) they were fixed in PBS containing 4% paraformaldehyde and 4% sucrose for 8 min. Duolink In Situ PLA Mouse/Rabbit kit with red detection reagents (DUO92101-1KT; Sigma-Aldrich) was used according to themanufacturer’s protocol.We used dynein intermediate chain antibody (MouseMAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit, 5519; Cell Signalling), or no second 1° antibody (negative control).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Membrane, Expressing

Journal: Molecules

Article Title: Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

doi: 10.3390/molecules26123732

Figure Lengend Snippet: Expression of the TAK1, JIP4, and Septin-2 proteins in ZIKV-infected and mock-infected cells and in cells with expression of active and inactive NS2B-NS3 protease. ( A ) U251 (upper panel), A549 (middle panel), and 293T (bottom panel) cells were infected with ZIKV R103451 Human 2015 Honduras, PRVABC59 or H/PAN/2016 strains, respectively, or inoculated with mock and analyzed by Western blotting. The TAK1, JIP4, and Septin-2 proteins were detected with specific antibodies. ( B ) 293T (left panel) and A549 (right) cells expressing NS2B-NS3 WT or NS2B-NS3 S135A were analyzed. Cells were harvested at 48 h after transfection, lysed, and lysates were analyzed by Western blotting. Levels and profiles of expression of TAK1, JIP4, and Septin-2 proteins were compared in a cell with an expression of active and inactive. Protease expression was confirmed by the presence of NS3 protein in both cell lines (upper panel).

Article Snippet: Additionally, for TAK1, JIP4, and Septin-2 detection, membranes were incubated with rabbit anti-TAK1 antibody (1.5 μg/mL; Thermo Scientific), mouse anti-JIP4 antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-Septin-2 antibody (1:2000; Santa Cruz Biotechnology), respectively.

Techniques: Expressing, Infection, Western Blot, Transfection