rabbit antibody against full length human pex14 protein  (Bethyl)

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 1 – Schematic representation of PEX13 and PEX14 and their interactions with each other and with PEX5. The amino acid positions of the globular domains of Rattus Norvegicus PEX13 and PEX14 proteins are indicated (CC, Coiled coil; NTD, N-terminal domain of PEX14; SH3, Src Homology 3 domain). Putative transmembrane domains (TMDs; light blue cylinders) were predicted with PRALINE™ [83] and PHOBIUS [84] using an alignment of twenty protein sequences representative of eukaryotic evolution. The pentapeptide motifs of PEX5 [43] are represented in orange. Black lines indicate the epitopes recognized by some of the antibodies used in this work. The interactions between PEX13, PEX14 and PEX5 are indicated as red (yeast) or blue (mammals) arrows. The SH3 domain of yeast PEX13 interacts with a PXXP motif (residues 87-90) located between the NTD and TMD of PEX14 [61,85]. In mammals, the SH3 domain of PEX13 interacts with the NTD of PEX14 [41,63]. The region comprising amino acid residues 236-246 of yeast PEX13, which corresponds to a region in the mammalian peroxin located between TMD2 and TMD3, interacts with an undefined region (marked with “?”) of PEX14 [47]. The SH3 of PEX13 also interacts with the N-terminal half of PEX5 in yeast [36,37,85]. A similar interaction involving residues 219-403 of human PEX13 (which includes the SH3 domain) and the N-terminal half of PEX5 has been described [38]. Residues 1-135 of mammalian PEX13 were also reported to interact with pentapeptides motifs 2-4 of PEX5 [45]. The NTD of PEX14 interacts with pentapeptide motifs present in the N-terminal half of PEX5 both in mammals and yeast/fungi [43,45,49,71,86]. A C- terminal domain of yeast PEX14 (residues 235-341) has been shown to interact with PEX5 [49,71], an interaction involving the second pentapeptide motif of PEX5 [49] (Reviewed in [51,57,70]).

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques:

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 2 – PEX14 is a transmembrane protein with a Nin-Cout topology. A- Expression and purification of H6TEVPEX14 using IMAC. Culture aliquots collected before (lane NI) and after (lane I) protein induction, lysed cell extract (lane T) and the corresponding soluble (lane S) and insoluble protein fractions (lane P), the non-bound protein fraction from the Nickel-NTA beads (NB) and the purified protein () – H6TEVPEX14 (lane P14) were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and stained with Ponceau S. B- Proteoliposomes (Lipos) containing either PEX14 (left panel) or H6TEVPEX14 (H6PEX14; right panel) were treated or not with PK (400 µg/mL) in the presence or absence of TX-100, and analyzed by SDS-PAGE/Western blot. PEX14 was detected with antibodies against the full-length (FL) protein. Equivalent amounts of recombinant proteins (Lanes I; 130 ng) and the corresponding reconstituted proteins were loaded onto the gels. C- As in B, but using PEX14 that was subjected to the reconstitution protocol in the absence of lipids. D- As in B, but using antibodies directed to the full-length protein, the C-terminal residues 327-377 of human PEX14, and the N-terminal histidine-tag. E- PEX14 proteoliposomes were treated with PK (400 µg/mL) or trypsin (Try, 400 µg/mL) and analyzed by SDS-PAGE/Western blot using an antibody against the full-length protein. The ~46-kDa PEX14 fragment obtained after trypsin digestion is recognized by the anti-PEX14(327-377) antibody (data not shown). Identical results were obtained with H6TEVPEX14; in this case, the two fragments with ~20 and ~30 kDa are recognized by the anti-His antibody (data not shown), suggesting that the 30-kDa fragment is a partial proteolysis product. F- Proteoliposomes, PK-treated proteoliposomes, and PK alone were subjected to SDS- PAGE, blotted onto a Sequi-blot PVDF membrane and stained with Coomassie Brilliant Blue. PK- resistant fragments of PEX14 (, 40 kDa and ~20 kDa) and protein bands derived from PK itself (*)

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques: Expressing, Purification, SDS Page, Membrane, Staining, Western Blot, Recombinant, Derivative Assay

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 1 – Schematic representation of PEX13 and PEX14 and their interactions with each other and with PEX5. The amino acid positions of the globular domains of Rattus Norvegicus PEX13 and PEX14 proteins are indicated (CC, Coiled coil; NTD, N-terminal domain of PEX14; SH3, Src Homology 3 domain). Putative transmembrane domains (TMDs; light blue cylinders) were predicted with PRALINE™ [83] and PHOBIUS [84] using an alignment of twenty protein sequences representative of eukaryotic evolution. The pentapeptide motifs of PEX5 [43] are represented in orange. Black lines indicate the epitopes recognized by some of the antibodies used in this work. The interactions between PEX13, PEX14 and PEX5 are indicated as red (yeast) or blue (mammals) arrows. The SH3 domain of yeast PEX13 interacts with a PXXP motif (residues 87-90) located between the NTD and TMD of PEX14 [61,85]. In mammals, the SH3 domain of PEX13 interacts with the NTD of PEX14 [41,63]. The region comprising amino acid residues 236-246 of yeast PEX13, which corresponds to a region in the mammalian peroxin located between TMD2 and TMD3, interacts with an undefined region (marked with “?”) of PEX14 [47]. The SH3 of PEX13 also interacts with the N-terminal half of PEX5 in yeast [36,37,85]. A similar interaction involving residues 219-403 of human PEX13 (which includes the SH3 domain) and the N-terminal half of PEX5 has been described [38]. Residues 1-135 of mammalian PEX13 were also reported to interact with pentapeptides motifs 2-4 of PEX5 [45]. The NTD of PEX14 interacts with pentapeptide motifs present in the N-terminal half of PEX5 both in mammals and yeast/fungi [43,45,49,71,86]. A C- terminal domain of yeast PEX14 (residues 235-341) has been shown to interact with PEX5 [49,71], an interaction involving the second pentapeptide motif of PEX5 [49] (Reviewed in [51,57,70]).

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques:

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 2 – PEX14 is a transmembrane protein with a Nin-Cout topology. A- Expression and purification of H6TEVPEX14 using IMAC. Culture aliquots collected before (lane NI) and after (lane I) protein induction, lysed cell extract (lane T) and the corresponding soluble (lane S) and insoluble protein fractions (lane P), the non-bound protein fraction from the Nickel-NTA beads (NB) and the purified protein () – H6TEVPEX14 (lane P14) were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and stained with Ponceau S. B- Proteoliposomes (Lipos) containing either PEX14 (left panel) or H6TEVPEX14 (H6PEX14; right panel) were treated or not with PK (400 µg/mL) in the presence or absence of TX-100, and analyzed by SDS-PAGE/Western blot. PEX14 was detected with antibodies against the full-length (FL) protein. Equivalent amounts of recombinant proteins (Lanes I; 130 ng) and the corresponding reconstituted proteins were loaded onto the gels. C- As in B, but using PEX14 that was subjected to the reconstitution protocol in the absence of lipids. D- As in B, but using antibodies directed to the full-length protein, the C-terminal residues 327-377 of human PEX14, and the N-terminal histidine-tag. E- PEX14 proteoliposomes were treated with PK (400 µg/mL) or trypsin (Try, 400 µg/mL) and analyzed by SDS-PAGE/Western blot using an antibody against the full-length protein. The ~46-kDa PEX14 fragment obtained after trypsin digestion is recognized by the anti-PEX14(327-377) antibody (data not shown). Identical results were obtained with H6TEVPEX14; in this case, the two fragments with ~20 and ~30 kDa are recognized by the anti-His antibody (data not shown), suggesting that the 30-kDa fragment is a partial proteolysis product. F- Proteoliposomes, PK-treated proteoliposomes, and PK alone were subjected to SDS- PAGE, blotted onto a Sequi-blot PVDF membrane and stained with Coomassie Brilliant Blue. PK- resistant fragments of PEX14 (, 40 kDa and ~20 kDa) and protein bands derived from PK itself (*)

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques: Expressing, Purification, SDS Page, Membrane, Staining, Western Blot, Recombinant, Derivative Assay