rabbit anti dynein  (Proteintech)

Journal: Cells

Article Title: Loss of Dnah5 Downregulates Dync1h1 Expression, Causing Cortical Development Disorders and Congenital Hydrocephalus

doi: 10.3390/cells13221882

Figure Lengend Snippet: Antibodies used for immunofluorescence staining and Western blotting analysis.

Article Snippet: Dynein , 532.4 , Rabbit anti-dynein antibody (ABT266, Sigma-Aldrich, MERCK KGaA, Darmstadt, Germany) , WB; 1:1000 , Not applicable.

Techniques: Immunofluorescence, Staining, Western Blot, Molecular Weight

Journal: Communications Biology

Article Title: Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

doi: 10.1038/s42003-023-04621-8

Figure Lengend Snippet: a OPN3 expression (green) colocalized with HEK marker Pan-CK (red) in skin explant with immunofluorescence staining, without UVA (top) or with UVA irradiation (bottom). Nuclei were counterstained with DAPI. Masson-Fontana (MF) staining demonstrated melanin cap formation. Images were analyzed by brightfield/fluorescence microscopy. Scale bar = 20 µm. b After siRNA inhibited OPN3 irradiated without or with UVA, WB was used to analyze changes in OPN3, Dync1i1, and DCTN1 expression levels in HEK. WB analyses were normalized using β-tubulin as a loading control, and the relative protein level was quantified using Quantity One software. n = 3 independent experiments. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, **** P < 0.0001. c After siRNA inhibited OPN3 irradiated without or with UVA, RT-qPCR was used to analyze changes in OPN3 mRNA levels in HEK ( n = 3 independent experiments). The data were presented as mean ± SEM. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001. d After siRNA inhibited OPN3 irradiated without or with UVA, RT-qPCR was used to analyze changes in DCTN1 mRNA expression levels in HEK ( n = 3 independent experiments). Statistical significance was determined by one-ANOVA with post-test. *** P < 0.001. e After siRNA inhibited OPN3 irradiated without or with UVA, RT-qPCR was used to analyze changes in Dync1i1 mRNA expression levels in HEK ( n = 3 independent experiments). Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01.

Article Snippet: The primary antibodies were used as follows: Rabbit polyclonal anti-human OPN3 (1:1000, AB_2837240, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human DCTN1 (1:1000, AB_2838577, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human cytoplasmic dynein 1 intermediate chain 1 (Dync1i1, 1:1000, AB_2846296, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human calmodulin-dependent protein kinase II (CaMKII, 1:1000, ab126789, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CaMKII (1:1000, ab124880, Cambridge, UK), Rabbit polyclonal anti-human CREB (1:1000, ab32515, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CREB (phosphorylated at S133) (1:1000, ab220798, Abcam, Cambridge, UK), Rabbit polyclonal anti-human Akt (1:1000, ABP0059, Abbkine Scientific, Wuhan, China), Rabbit polyclonal anti-human p-Akt (phosphorylated at Ser473) (1:1000, ABP0030, Abbkine Scientific, Wuhan, China).

Techniques: Expressing, Marker, Immunofluorescence, Staining, Irradiation, Fluorescence, Microscopy, Software, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

doi: 10.1038/s42003-023-04621-8

Figure Lengend Snippet: a HaCaT was transfected with lentivirus shOPN3 (shOPN3#1) and control lentivirus (shNC), and after irradiated without or with UVA, WB was used to analyze changes of OPN3, Dync1i1, and DCTN1 protein expression levels in HaCaT ( n = 3 independent experiments). WB analyses were normalized using β-tubulin as a loading control, and the relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. b – d After shOPN3 inhibited OPN3 irradiated without or with UVA, RT-qPCR was used to analyze changes of OPN3, Dync1i1, and DCTN1 mRNA expression in HaCaT ( n = 3 independent experiments). Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. e HaCaT was transfected with lentivirus overexpression OPN3 (LV-OPN3) and control lentivirus (LV-control) and irradiated without or with UVA. Then, WB was used to analyze changes in OPN3, Dync1i1, and DCTN1 expression in HaCaT. β-tubulin was used as a loading control. Statistical significance was determined by one-ANOVA with post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. f – h After LV-OPN3 overexpression of OPN3 irradiated without or with UVA, RT-qPCR was used to analyze changes in OPN3, Dync1i1, and DCTN1 expression levels in HaCaT. OPN3, Dync1i1, and DCTN1 mRNA levels were normalized to GAPDH levels ( n = 3 independent experiments). Statistical significance was determined by one-ANOVA with post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies were used as follows: Rabbit polyclonal anti-human OPN3 (1:1000, AB_2837240, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human DCTN1 (1:1000, AB_2838577, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human cytoplasmic dynein 1 intermediate chain 1 (Dync1i1, 1:1000, AB_2846296, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human calmodulin-dependent protein kinase II (CaMKII, 1:1000, ab126789, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CaMKII (1:1000, ab124880, Cambridge, UK), Rabbit polyclonal anti-human CREB (1:1000, ab32515, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CREB (phosphorylated at S133) (1:1000, ab220798, Abcam, Cambridge, UK), Rabbit polyclonal anti-human Akt (1:1000, ABP0059, Abbkine Scientific, Wuhan, China), Rabbit polyclonal anti-human p-Akt (phosphorylated at Ser473) (1:1000, ABP0030, Abbkine Scientific, Wuhan, China).

Techniques: Transfection, Irradiation, Expressing, Software, Quantitative RT-PCR, Over Expression

Journal: Communications Biology

Article Title: Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

doi: 10.1038/s42003-023-04621-8

Figure Lengend Snippet: a HEK treated with PTX was stimulated with 3 J cm −2 UVA, and calcium flux was quantified by flow cytometry. n = 3 independent experiments. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001. b HEK treated with PTX were stimulated with 3 J cm −2 UVA, and p-CaMKII and p-CREB protein expression level was analyzed by WB. WB analyses were normalized using β-tubulin as a loading control, and the relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001. c HEK were irradiated without and with UVA, and PLC-β protein levels were determined by WB. d HEK treated with PTX were stimulated with 3 J cm −2 UVA; protein level was analyzed by WB. e HEK were transfected with siRNA against OPN3 irradiated with UVA and lysed after 1 h. Lysates were analyzed by WB using the indicated antibodies (anti-OPN3 and anti-PLC-β). β-tubulin was used as a loading control. f HEK treated with U73122 were stimulated with 3 J cm −2 UVA; calcium flux was quantified by flow cytometry. n = 3 independent experiments. Statistical significance was determined by one-ANOVA with post-test. *** P < 0.001, **** P < 0.0001. g HEK treated with U73122 were stimulated with 3 J cm −2 UVA; p-CaMKII, p-CREB, DCTN1, and Dync1i1 protein expression level was analyzed by WB. WB analyses were normalized using β-tubulin as a loading control, and the relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies were used as follows: Rabbit polyclonal anti-human OPN3 (1:1000, AB_2837240, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human DCTN1 (1:1000, AB_2838577, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human cytoplasmic dynein 1 intermediate chain 1 (Dync1i1, 1:1000, AB_2846296, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human calmodulin-dependent protein kinase II (CaMKII, 1:1000, ab126789, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CaMKII (1:1000, ab124880, Cambridge, UK), Rabbit polyclonal anti-human CREB (1:1000, ab32515, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CREB (phosphorylated at S133) (1:1000, ab220798, Abcam, Cambridge, UK), Rabbit polyclonal anti-human Akt (1:1000, ABP0059, Abbkine Scientific, Wuhan, China), Rabbit polyclonal anti-human p-Akt (phosphorylated at Ser473) (1:1000, ABP0030, Abbkine Scientific, Wuhan, China).

Techniques: Flow Cytometry, Expressing, Software, Irradiation, Transfection

Journal: Communications Biology

Article Title: Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

doi: 10.1038/s42003-023-04621-8

Figure Lengend Snippet: a Cells were irradiated without and with UVA, and AKT protein and phosphorylated-AKT (p-AKT) protein levels were determined by WB. β-tubulin was used as a loading control. The relative protein level was quantified using Quantity One software. Statistical significance was determined by t -test analysis. ** P < 0.01. b HEK were transfected with siRNA against OPN3 irradiated with UVA and lysed after 1 h. Lysates were analyzed by WB using the indicated antibodies (anti-OPN3, anti-AKT, anti-p-AKT, anti-Dync1i1, and anti-DCTN1). β-tubulin was used as a loading control. The relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. *** P < 0.001. c HEK treated with U73122 were stimulated with 3 J/cm 2 UVA, and protein lysate was analyzed by WB. β-tubulin was used as a loading control. The relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. *** P < 0.001. d HEK treated with MK-2206 (2HCl) were stimulated with 3 J cm −2 UVA, and protein lysate was analyzed by WB. β-tubulin was used as a loading control. The relative protein level was quantified using Quantity One software. Statistical significance was determined by one-ANOVA with post-test. ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies were used as follows: Rabbit polyclonal anti-human OPN3 (1:1000, AB_2837240, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human DCTN1 (1:1000, AB_2838577, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human cytoplasmic dynein 1 intermediate chain 1 (Dync1i1, 1:1000, AB_2846296, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human calmodulin-dependent protein kinase II (CaMKII, 1:1000, ab126789, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CaMKII (1:1000, ab124880, Cambridge, UK), Rabbit polyclonal anti-human CREB (1:1000, ab32515, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CREB (phosphorylated at S133) (1:1000, ab220798, Abcam, Cambridge, UK), Rabbit polyclonal anti-human Akt (1:1000, ABP0059, Abbkine Scientific, Wuhan, China), Rabbit polyclonal anti-human p-Akt (phosphorylated at Ser473) (1:1000, ABP0030, Abbkine Scientific, Wuhan, China).

Techniques: Irradiation, Software, Transfection

Journal: Communications Biology

Article Title: Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

doi: 10.1038/s42003-023-04621-8

Figure Lengend Snippet: OPN3 upregulates expression of Dync1i1 and DCTN1 in keratinocytes after UVA radiation exposure via the calcium-dependent G protein-coupled and Akt signaling pathway. (This figure was created based on the elements in the Science Slides software ( http://scienceslides.com/ ).

Article Snippet: The primary antibodies were used as follows: Rabbit polyclonal anti-human OPN3 (1:1000, AB_2837240, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human DCTN1 (1:1000, AB_2838577, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human cytoplasmic dynein 1 intermediate chain 1 (Dync1i1, 1:1000, AB_2846296, Affinity Biosciences Ltd., Beijing, China), Rabbit polyclonal anti-human calmodulin-dependent protein kinase II (CaMKII, 1:1000, ab126789, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CaMKII (1:1000, ab124880, Cambridge, UK), Rabbit polyclonal anti-human CREB (1:1000, ab32515, Abcam, Cambridge, UK), Rabbit polyclonal anti-human p-CREB (phosphorylated at S133) (1:1000, ab220798, Abcam, Cambridge, UK), Rabbit polyclonal anti-human Akt (1:1000, ABP0059, Abbkine Scientific, Wuhan, China), Rabbit polyclonal anti-human p-Akt (phosphorylated at Ser473) (1:1000, ABP0030, Abbkine Scientific, Wuhan, China).

Techniques: Expressing, Software

Journal: bioRxiv

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

doi: 10.1101/2023.01.28.526044

Figure Lengend Snippet: (A) Schematic illustrating our single-molecule motility assay. (B) Example kymographs showing the growth and catastrophe dynamics used to differentiate the plus-end of the microtubule from the more stable minus-end. (C) Quantification of the directionality of runs on each microtubule. Runs were defined as events ≥ 0.8 µm in length towards either the minus- or plus-end of the microtubule (MT). Symbols indicate comparison to the BICD2N dynein positive control. Kruskal-Wallis test with Dunn’s multiple comparisons. n = 20 MT each. K560 v. BICD2N, P < 0.0001; K560 v. JIP3, P < 0.0001; K560 v. JIP4, P < 0.0001; BICD2N v. JIP3, P > 0.9999; BICD2N v. JIP4, P > 0.9999; JIP3 v. JIP4, P > 0.9999. (D-F) Example kymograph and quantification showing the activity of BICD2N-containing dynein complexes. (G-I) Example kymograph and quantification showing the activity of JIP3-containing dynein complexes. (J-L) Example kymograph and quantification showing the activity of JIP4-containing dynein complexes. All velocity histograms were fit to a Gaussian curve and all run length histograms (1– cumulative distribution frequency) were fit to a one phase decay. Listed values are median (25 th percentile-75 th percentile). n = 97-192 events. Complexes with a net direction of “0” were stationary landing events, while complexes with a net direction of “–” or “+” moved ≥ 0.8 µm towards the minus- or plus-end of the microtubule respectively. n = 20 MT each; Kruskal-Wallis test with Dunn’s multiple comparisons; JIP3 (0 v. –, P = 0.6412; 0 v. +, P = 0.0051; – v. +, P < 0.0001); JIP4 (0 v. –, P > 0.9999; 0 v. +, P = 0.0004; – v. +, P < 0.0001).

Article Snippet: We used dynein intermediate chain antibody (Mouse MAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit Cell Signalling, 5519), or no second 1° antibody (negative control).

Techniques: Motility Assay, Comparison, Positive Control, Activity Assay

Journal: bioRxiv

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

doi: 10.1101/2023.01.28.526044

Figure Lengend Snippet: (A) We demonstrate that the GTP-bound ARF6 is enriched on AV membranes, where it can recruit its interacting partners JIP3 or JIP4. JIP3/4 can then recruit dynactin and dynein and activate minus-end-directed retrograde motility especially of autolysosomes. By contrast, we propose that ARF6 is removed from the lysosomal membrane by local ARF6 GAP activity, which may be promoted by the presence of phosphorylated RABs, including RAB10. RAB10 plays an unknown role in transport, but seems to induce anterograde transit, possibly through a JIP3- or JIP4-JIP1-kinesin-1 complex. These motor complexes are not the only ones involved in AV or lysosome transport; we highlight a few complementary complexes on the left. (B) One mechanism by which this pathway may be disrupted in neurodegeneration is via hyperphosphorylation of RABs. The disease-causing mutations in LRRK2 kinase result in increased phospho-RABs and also increased recruitment of kinesin-1 to the AV membrane. However, the resulting loss of AV motility can be rescued by expressing GTP-locked ARF6; thus these motor-regulatory mechanisms are interconnected and possibly competitive.

Article Snippet: We used dynein intermediate chain antibody (Mouse MAB1618) plus JIP3 antibody (Rabbit ab196761), JIP4 antibody (Rabbit Cell Signalling, 5519), or no second 1° antibody (negative control).

Techniques: Membrane, Activity Assay, Expressing