Western blot analysis of A549 human alveolar adenocarcinoma whole cell lysate probed with Mouse anti Human PGP9.5 antibody (7863-1004) followed by HRP conjugated Goat anti Mouse IgG, visualized by chemiluminescence

Published investigator image:
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
UCH-L1 exists in both cytosolic and membrane-associated forms in neurons. Cell lines contain much lower levels of membrane-associated UCH-L1 than neurons. A, UCH-L1 exists in cytosolic and membrane-enriched fractions of whole rat brain. Adult male rat brain was homogenized and subjected to subcellular fractionation. Fractions were immunoblotted for protein markers to verify enrichment. 30 µg of protein was loaded in each lane. Numbers shown are in kilodaltons relative to molecular weight standards. P1, nuclear-enriched fraction; P2, crude membranes and synaptosomes; P3, microsomes, including the endoplasmic reticulum and Golgi; S2, cytosol and microsomes; S3, cytosol; Mito, mitochondria; Syn, synaptosomes. B, UCH-L1 exists in cytosolic and membrane fractions of cultured cortical neurons. Rat cortical neuronal lysate was subjected to high-speed ultracentrifugation to isolate the soluble cytosolic supernatant and membrane pellet fractions. Protein distribution was tested by immunoblotting for soluble cytosolic protein (RhoGDI) and transmembrane protein (GluA1). A proportion of Rac1 in neurons is peripherally associated with the membrane via geranylgeranylation, and the endogenous distribution is preserved using this protocol. 15 µg of protein was loaded in each lane. C, relative proportions of UCH-L1 detected per microgram of protein in the cytosolic and membrane fractions of cultured cortical neurons. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software. a.u., arbitrary units. D, cell lines contain much lower levels of UCH-L1M than cultured cortical neurons. Lysates from the indicated clonal cell lines were subjected to high-speed ultracentrifugation to isolate the cytosolic and membrane fractions. Protein distribution was tested by immunoblotting for cytosolic protein (RhoGDI), and prenylated protein (Rac1). The distribution of Rac1 is preserved in cell lines, although the proportions vary between cell lines. UCH-L1 was not detected in the membrane fraction of COS7 or HEK293T cells and only to a minor extent with increased gain in N2a cells. 20 µg of protein was loaded in each lane. E, relative proportions of UCH-L1 detected per microgram of protein in the cytosolic and membrane fractions of the indicated cell lines. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software. F, serum starvation induces outgrowth of neuronal-like processes in N2a cells. GFP-transfected N2a cells were exposed to serum starvation for 24 h and fixed for confocal microscopy. Cell outlines were identified, and images were prepared using ImageJ software with MacBiophotonics plug-ins. G, differentiation does not alter the proportion of UCH-L1M in N2a cells. Outgrowth of neuronal-like processes was induced in N2a cells with 24-h serum starvation, and then cells were subjected to ultracentrifugation as before. The proportion of UCH-L1M remained unchanged compared with untreated N2a cells. H, relative proportions of UCH-L1M detected per microgram of protein in the membrane fraction of N2a cells. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software.

From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.

Published investigator image:
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
The farnesyltransferase inhibitor FTI-276 does not reduce UCH-L1M in neurons. A, membrane-associated H-ras, but not UCH-L1, is decreased by farnesyltransferase inhibition. Cultured cortical neurons were treated with a DMSO control or increasing amounts of the farnesyltransferase inhibitor FTI-276 (Calbiochem), as indicated, for 48 h and then lysed and subjected to high-speed ultracentrifugation to separate the cytosolic and membrane fractions. Immunoblotting for the known farnesylation substrate H-ras shows an increasing shift from a membrane-associated form to a cytosolic form as the concentration of FTI-276 increases, whereas the proportion of UCH-L1 remains unaffected. B, relative proportions of UCH-L1M and H-rasM detected per microgram of protein in the FTI-treated membrane fractions relative to the DMSO control (n = 6 for the DMSO control and 500 nm, and n = 5 for both 1 and 3 µm). Data were analyzed using one-way analysis of variance with post hoc Bonferroni test. *, p < 0.05; **, p < 0.001. a.u., arbitrary units.

From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.

Published investigator image:
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
Selected mutations do not reduce membrane-associated UCH-L1M. A, three-dimensional models showing the location of visible mutated or deleted residues in UCH-L1 using Cn3D software with UCH-L1 PDB codes 2ETL and 3KW5 (bound to UbVME). The Ile-93 residue is buried internally. B, effect of individual UCH-L1 cysteine mutations on UbVME binding in N2a cells. N2a cells were transfected with HA-UCH-L1 Cys-to-Ser point mutants as indicated. Cell lysates were incubated with 2 µm UbVME to assess the effect of mutation on substrate binding and then immunoblotted (IB) to detect infected HA-UCH-L1 constructs and total (endogenous + infected) UCH-L1. C, 3CS (C90S, C152S, and C220S) and D30K mutations abolish UbVME binding, but I93M does not. Cultured cortical neurons were infected with Sindbis virus expressing the indicated HA-tagged UCH-L1 constructs. Lysates were incubated with 2 µm UbVME to assess the effect of mutations on substrate binding and then immunoblotted to detect infected HA-UCH-L1 constructs and total (endogenous and infected) UCH-L1. D, 3CS (C90S, C152S, and C220S), D30K, and I93M mutations do not reduce the proportion of UCH-L1M. Cultured cortical neurons were infected with Sindbis virus expressing the indicated HA-tagged UCH-L1. Lysates were subjected to ultracentrifugation to separate cytosolic and membrane-associated proteins. The fractions were immunoblotted to detect infected HA-UCH-L1 constructs and total (endogenous and infected) UCH-L1. E, relative proportions of the indicated HA-UCH-L1M constructs detected per microgram of protein in the membrane fraction relative to the cytosolic fraction (WT, n = 9; 3CS, n = 3; D30K, n = 5; I93M, n = 3). a.u., arbitrary units.

From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.

Published investigator image:
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
Deletion of the final four residues of UCH-L1 leads to increased cell death. A, deletion of the final four residues leads to altered UCH-L1 distribution. Hippocampal neurons were infected with Sindbis virus expressing the indicated HA-UCH-L1 constructs to assess the distribution of total (endogenous and infected) UCH-L1 and infected HA-UCH-L1. HA-UCH-L1 distribution in WT, N-terminal truncation, and CTT?2-infected cells is comparable with endogenous UCH-L1. Infected protein is largely absent from the processes. B, deletion of the final four residues leads to increased cell death. The cell death assay measured the release of lactate dehydrogenase. Infection with Sindbis virus expressing the HA-CTT?4 construct caused a significant increase in cell death in cortical neurons compared with HA-WT. Control, uninfected. Data were analyzed using one-way analysis of variance with post hoc Bonferroni test. *, p < 0.05. Control, HA-WT, and HA-CTT?4, n = 5; HA-NTT, n = 4; HA-CTT?2, n = 3.

From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.