- PA5-25584 - Invitrogen Antibodies MARCHF5 antibody

Submitted references Parkin is a disease modifier in the mutant SOD1 mouse model of ALS.

Palomo GM, Granatiero V, Kawamata H, Konrad C, Kim M, Arreguin AJ, Zhao D, Milner TA, Manfredi G
EMBO molecular medicine 2018 Oct;10(10)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis using a MARCH5 polyclonal antibody (Product # PA5-25584) in mouse kidney tissue lysates (35 µg per lane).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of MARCH5 was achieved by transfecting HeLa with MARCH5 specific siRNAs (Silencer® select Product # s29332 and s29333). Western blot analysis (Fig. a) was performed using whole cell extracts from the MARCH5 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MARCH5 Polyclonal Antibody (Product # PA5-25584, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000). 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 MARCH5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using MARCH5 Polyclonal Antibody (Product # PA5-25584) and a 28 kDa band corresponding to MARCH5 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), T-47D (Lane 2), MCF 10A (Lane 3), A549 (Lane 4), HeLa (Lane 5), PC-3 (Lane 6) and HCT 116 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis in formalin-fixed, paraffin-embedded human skeletal muscle using a MARCH5 polyclonal antibody (Product # PA5-25584), followed by HRP-conjugated secondary antibody and DAB staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of MCF-7 cells using a MARCH5 polyclonal antibody (Product # PA5-25584) (right) compared to a negative control cell (left) at a dilution of 1:10-50, followed by a FITC-conjugated goat anti-rabbit antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 Parkin knockout improves the decline of mitochondrial dynamics proteins in SOD 1-G93A spinal cord, but mitochondrial protein ubiquitination profiles and March5 and Mul1 levels are unaffected A, B Western blots (A) and quantification (B) of Miro1 in spinal cord homogenates at disease end stage. beta-actin is used as loading reference. Results are expressed as mean +- SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group; * P = 0.011 by paired Student's t -test (for comparison G93A vs. PKO/G93A) and ** P = 0.003 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A). Parkin knockout increases the levels of Miro1 in SOD1-G93A mice at disease end stage. C, D Western blots (C) and quantification (D) of Mfn2 in spinal cord homogenates at disease end stage. Protein levels are normalized by beta-actin. Results are expressed as mean +- SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.078 by paired Wilcoxon's test); *** P = 0.0007 by paired Friedman's test with Dunn's correction (for Non Tg vs. G93A) and * P = 0.037 by paired Friedman's test with Dunn's correction (for PKO vs. PKO/G93A). Parkin knockout increases the levels of Mfn2 in SOD1-G93A mice at disease end stage. E Western blots of spinal cord mitochondria at disease end stage probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. C

PA5-25584

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