- PA1-523 - Invitrogen Antibodies ARNTL antibody

Submitted references Prolonged Administration of Melatonin Ameliorates Liver Phenotypes in Cholestatic Murine Model.

Ceci L, Chen L, Baiocchi L, Wu N, Kennedy L, Carpino G, Kyritsi K, Zhou T, Owen T, Kundu D, Sybenga A, Isidan A, Ekser B, Franchitto A, Onori P, Gaudio E, Mancinelli R, Francis H, Alpini G, Glaser S
Cellular and molecular gastroenterology and hepatology 2022;14(4):877-904

Melatonin modulates daytime-dependent synaptic plasticity and learning efficiency.

Jilg A, Bechstein P, Saade A, Dick M, Li TX, Tosini G, Rami A, Zemmar A, Stehle JH
Journal of pineal research 2019 Apr;66(3):e12553

Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes.

Akagi R, Akatsu Y, Fisch KM, Alvarez-Garcia O, Teramura T, Muramatsu Y, Saito M, Sasho T, Su AI, Lotz MK
Osteoarthritis and cartilage 2017 Jun;25(6):943-951

The transcription factor Runx2 is under circadian control in the suprachiasmatic nucleus and functions in the control of rhythmic behavior.

Reale ME, Webb IC, Wang X, Baltazar RM, Coolen LM, Lehman MN
PloS one 2013;8(1):e54317

Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1-7 neurons.

Gillespie JM, Chan BP, Roy D, Cai F, Belsham DD
Endocrinology 2003 Dec;144(12):5285-92

Circadian Transcription. Thinking outside the E-Box.

Muñoz E, Brewer M, Baler R
The Journal of biological chemistry 2002 Sep 27;277(39):36009-17

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of BMAL1 was performed by loading 25 µg of U251 (Lane 1), U87-MG (Lane 2), and NIH-3T3 cell lysates (Lane 3) and a molecular weight protein ladder onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with a blocking buffer at 4ºC overnight. The membrane was probed with a BMAL1 polyclonal antibody (Product # PA1-523) at a dilution of 1:500 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at 69 kDa in all three cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), U-87 MG (Lane 2), U-2 OS (Lane 3), NIH/3T3 (Lane 4), HeLa (Lane 5), HEK 293 (Lane 6), Rat Brain (tissue extract) (Lane 7) and Jurkat (Lane 8). The blot was probed with Anti-BMAL 1 Rabbit Polyclonal Antibody (Product # PA1-523, 1:100-1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Three isoforms of 68, 55, 25 kDa corresponding to BMAL 1 were observed across the cell lines and tissue tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of BMAL1 was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with BMAL1 Rabbit Polyclonal Antibody (Product # PA1-523) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of BMAL1 was done on SH-SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with BMAL1 Rabbit Polyclonal Antibody (PA1-523, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10, 000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Melatonin influences the phenotype of isolated P92-PSC. ( A ) Morphologic shapes of human isolated PSC cell line. Scale bars : 20 mum. ( B ) By immunofluorescence and qPCR, the MT1 and ( C and D ) clock genes (PER1, CRY1, CLOCK, and BMAL1) are decreased in isolated PSC cell lines treated with melatonin compared with vehicle-treated PSC cells. Original magnification, 20x; scale bars : 20 mum. Data are means +- SEM of 2 evaluations from 3 cumulative preparations of in vitro experiments. Each dot represents 1 value in data set. * P < .05 vs PSC.

PA1-523

Antibody data