- PA5-42646 - Invitrogen Antibodies RHOT1 antibody

Submitted references A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1.

Fagbadebo FO, Kaiser PD, Zittlau K, Bartlick N, Wagner TR, Froehlich T, Jarjour G, Nueske S, Scholz A, Traenkle B, Macek B, Rothbauer U
Frontiers in molecular biosciences 2022;9:835302

Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro: The role of Miro1.

Tseng N, Lambie SC, Huynh CQ, Sanford B, Patel M, Herson PS, Ormond DR
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 2021 Apr;41(4):761-770

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of human A549 cells using an anti-RHOT1 polyclonal antibody (Product # PA5-42646).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of mouse brain and human neuroblastoma cells using an anti-RHOT1 polyclonal antibody (Product # PA5-42646).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched cell extracts (30 µg lysate) of HEK293 (Lane 1), PANC1 (Lane 2), HT29 (Lane 3), NK-92 (Lane 4), tissue extracts of Mouse Brain (Lane 5) and Rat Brain (Lane 6). The blot was probed with Anti-RHOT1 Polyclonal Antibody (Product # PA5-42646, 1µg/ml) 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 70 kDa band corresponding to RHOT1 was observed across all the cell lines and tissues. A non-specific band was observed at 35 kDa for all cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of human fetal lung cell lysate using an anti-RHOT1 polyclonal antibody (Product # PA5-42646).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of RHOT1 was achieved by transfecting HEK 293 cells with RHOT1 specific siRNAs (Silencer® select Product # s30648). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the RHOT1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RHOT1 Polyclonal Antibody (Product # PA5-42646, 1µg/ml) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/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 RHOT1. [Note: Non-specific band observed at ~35Kda].

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (paraffin-embedded) analysis of human muscle tissue using an anti-RHOT1 polyclonal antibody (Product # PA5-42646).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 2 Immunoprecipitation of Miro1 with Nbs. (A) For immunoprecipitation with immobilized Nbs (nanotraps), soluble protein fraction of HEK293 cells transiently expressing GFP-Miro1 or GFP as control, was adjusted to 2 mg/ml and incubated with equal amounts of nanotraps. Input (IN, 1% of total) and bound (20% of total) fractions were subjected to SDS-PAGE followed by immunoblot analysis using antibodies specific for GFP (upper panel) and GAPDH (lower panel). As positive control GFP-nanotrap and as negative a non-specific (NC) nanotrap were used. (B) Immunoprecipitation from non-transfected HEK293 as described in (A) were performed. Input and bound fractions were analysed with an anti-Miro1 antibody. As positive control anti-Miro1 IgG immobilized on Protein A/G sepharose and as negative control a non-specific (NC) nanotrap was used. For densitometric evaluation immunoblot signals of endogenous Miro1 in the corresponding bound fractions were normalized to the Miro1 signal in the input, which was set to 1. Shown are the mean signals from three independent biological replicates +-SD. (C) Immunofluorescence (IF) detection of GFP-Miro1 in fixed and permeabilized HeLa cells after staining with Miro1-Nbs as primary labelling probes. Representative confocal laser scanning (CLSM) images are shown of each individual Nb detected with anti-VHH antibody labelled with Cy5 (middle row). As positive control, transfected cells were stained with anti-Miro1 antibody followed by detection with

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 5 Proteomic analysis of Miro1 capture. (A) For comparable immunoprecipitation soluble protein fraction of HEK293 cells were incubated either with monovalent Nbs either chemically coupled to NHS sepharose (M41 NHS , M114 NHS ) or the bivalent formats, which were site specifically conjugated to agarose particles by sortagging and click chemistry (bivM41 sort , bivM114 sort ). Input and bound fractions were analysed with an anti-Miro1 antibody. As positive control anti-Miro1 IgG immobilized on Protein A/G sepharose and as negative control a non-specific bivalent and site specifically conjugated nanotrap (bivNC sort ) was used. Shown is a representative immunoblot stained with an anti-Miro1 antibody. For densitometric evaluation immunoblot signals of endogenous Miro1 in the corresponding bound fractions were normalized to the Miro1 signal in the input, which was set to 1. Shown are the mean signals from three independent biological replicates +-SD. (B) Capture efficiency by mono- and bivalent nanotraps. Averaged iBAQ (intensity based absolute quantification) values for Miro1 isoform 2 (white circles) and isoform 3 (black circles) of three biological replicates are shown. (C) Classification of Miro1 interactor based on STRING database. Class 1: direct interactor, confidence score >0.9; Class 2: direct interactor, confidence score 0.9.

PA5-42646

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