- PA5-28185 - Invitrogen Antibodies MAD1L1 antibody

Submitted references Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions.

Kumari A, Kumar C, Pergu R, Kumar M, Mahale SP, Wasnik N, Mylavarapu SVS
The Journal of cell biology 2021 Dec 6;220(12)

The dynein adaptor Hook2 plays essential roles in mitotic progression and cytokinesis.

Dwivedi D, Kumari A, Rathi S, Mylavarapu SVS, Sharma M
The Journal of cell biology 2019 Mar 4;218(3):871-894

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: MAD1 Polyclonal Antibody detects MAD1L1 protein by western blot analysis. A. 30 µg 293T whole cell lysate/extract. B. 30 µg A431 whole cell lysate/extract. C. 30 µg HeLa whole cell lysate/extract. D. 30 µg HepG2 whole cell lysate/extract. E. 30 µg A375 whole cell lysate/extract.7.5% SDS-PAGE. MAD1 Polyclonal Antibody (Product # PA5-28185) dilution: 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of MAD1 was achieved by transfecting HCT 116 cells with MAD1 specific siRNAs (Silencer® select Product # s228313, s15906). Western blot analysis (Fig. a) was performed using whole cell extracts (1% SDS) from the MAD1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with MAD1 Polyclonal Antibody (Product # PA5-28185, 1:4000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to MAD1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on modified whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), MCF-7 (Lane 2), MDA-MB-231 (Lane 3), SK-BR-3 (Lane 4), KARPAS 299 (Lane 5) and HT-29 (Lane 6). The blot was probed with MAD1 Polyclonal Antibody (Product # PA5-28185, 1:2000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 80kDa band corresponding to MAD1 was observed across the panel

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MAD1 in paraformaldehyde-fixed MCF-7 cells using a MAD1 polyclonal antibody (Product # PA5-28185) at a 1:500 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of MAD1 was performed using 70% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MAD1 Polyclonal Antibody (Product # PA5-28185) at 5 µg/mL concentration in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded HBL435 xenograft, using MAD1 (Product # PA5-28185) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of MAD1 was performed in HeLa whole cell extracts using 5 µg of MAD1 Polyclonal Antibody (Product # PA5-28185). Samples were transferred to a membrane and probed with MAD1 Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure S4. LIC1-CTD phosphorylation is required for metaphase inactivation of the SAC in HeLa cells. (A) Graph depicting quantification of the metaphase index upon rescue of siRNA-mediated hLIC1 depletion by the various phosphomutant rLIC1 constructs, in the presence (no Mad2 siRNA) and absence (Mad2 siRNA) of a functional SAC. Mock and LIC1 bars are the same data as shown in Fig. S2 C . P values for each of the phosphomutant constructs (sixth bar from the left and beyond) have been calculated with respect to SST. n = 3, >=500 cells per experiment. (B) Immunoblots (IB) depicting the siRNA-mediated depletion of Mad2 and LIC1 in HeLa cells under the indicated conditions. (C and E) Representative confocal micrographs of congressed metaphase HeLa cells (released from nocodazole into MG132) immunostained to visualize the chromosomes (DAPI, blue), kinetochores (CREST, red), and the indicated SAC proteins, Zw10 or Mad1 (green). All cells were treated with anti-hLIC1 siRNA and rescued by the expression of the indicated rLIC1-CTD constructs. n = 3 independent experiments, >=30 metaphase cells per condition. Scale bar = 10 um. (D and F) Scatterplots depicting the quantification of the immunofluorescence signals of SAC proteins Zw10 (D) and Mad1 (F) normalized to the respective kinetochore (CREST) intensities under the indicated conditions. Kinetochore intensities from >=10 perfectly aligned metaphase cells per experiment over 3 independent experiments were quantified. (G) Representativ

PA5-28185

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