rabbit anti dctn1  (Proteintech)

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