Journal: Molecular Biology of the Cell

Article Title: GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

doi: 10.1091/mbc.E12-07-0525

Figure Lengend Snippet: Detection of endogenous GOLPH3 and GOLPH3L. (A) Pan-specific GOLPH3 antibody detects GOLPH3 and GOLPH3L equally well. EGFP-GOLPH3 (G3) and EGFP-GOLPH3L (G3L) expressed at similar levels in HeLa cells, as shown by EGFP Western blot, are detected equally well by antibody raised against GOLPH3, as shown by GOLPH3 Western blot. (B) Endogenous GOLPH3 and GOLPH3L are detected by pan-specific GOLPH3 antibody. SDS lysates from HEK 293 cells, each sample transfected with a combination of two of the following siRNA oligos: control siRNA, siRNA specific to GOLPH3, and each of three siRNAs specific to GOLPH3L. All samples were Western blotted using pan-specific GOLPH3 antibody and GAPDH antibody as a loading control. Lysates from control siRNA–treated cells are loaded at different relative amounts to provide a standard curve to allow semiquantitative assessment of knockdown efficiency. The upper band detected by pan-specific GOLPH3 antibody (∼34 kDa) corresponds to the predicted molecular weight of GOLPH3 and is specifically depleted when cells are transfected with siRNA specific to GOLPH3. The lower band (∼33 kDa) corresponds to the predicted molecular weight of GOLPH3L and is specifically depleted when cells are transfected with each of three siRNAs specific to GOLPH3L. Combined transfection of siRNAs to GOLPH3 and GOLPH3L results in depletion of both proteins.

Article Snippet: Mammalian cell lines, including HEK 293 (AD-293 variant; Stratagene, Santa Clara, CA), NIH 3T3 fibroblasts, HeLa S3, LNCaP, MCF-7, Neuro2A, NRK, N-Tera 2, RAW macrophages, and U2-OS cells, were grown according to American Type Culture Collection (Manassas, VA) guidelines.

Techniques: Western Blot, Transfection, Molecular Weight

Journal: Molecular Biology of the Cell

Article Title: GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

doi: 10.1091/mbc.E12-07-0525

Figure Lengend Snippet: Overexpression of GOLPH3L causes Golgi compaction. HEK 293 cells were transiently transfected with expression vectors for GOLPH3, GOLPH3L, or empty vector and cotransfected with one-fifth as much expression vector for EGFP to mark transfected cells. (A) Demonstration by Western blot of overexpression of GOLPH3 or GOLPH3L together with EGFP in HEK 293 cells. Duplicate transfections are shown. Western blot to GAPDH provides an indication of relative total protein loading. (B) Representative IF images of cells overexpressing GOLPH3 or GOLPH3L compared with control. Transfected cells are detected by expression of cotransfected EGFP, shown in green. DAPI marks the nucleus, in blue. GOLPH3 IF is shown in red, and increased GOLPH3 staining is evident in GOLPH3-overexpressing cells. TGN46 staining, shown in magenta, marks the location of the Golgi. Compared to cells transfected with empty vector, GOLPH3-overexpressing cells have a more expanded Golgi, whereas GOLPH3L-overexpressing cells have a more compact Golgi. Bar, 10 μm. (C) Quantification of Golgi area of cells from B. The Golgi area for each cell was measured, normalized to the nuclear area, and expressed relative to control. GOLPH3 overexpression results in expansion of the Golgi compared with control cells. Conversely, GOLPH3L overexpression leads to a significant compaction of the Golgi compared with control cells. Graphed are mean and SEM. The number of cells measured (pooled from three independent experiments) and p values vs. the empty vector control (by unpaired t test) are indicated.

Article Snippet: Mammalian cell lines, including HEK 293 (AD-293 variant; Stratagene, Santa Clara, CA), NIH 3T3 fibroblasts, HeLa S3, LNCaP, MCF-7, Neuro2A, NRK, N-Tera 2, RAW macrophages, and U2-OS cells, were grown according to American Type Culture Collection (Manassas, VA) guidelines.

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: Molecular Biology of the Cell

Article Title: GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

doi: 10.1091/mbc.E12-07-0525

Figure Lengend Snippet: GOLPH3 family members are dispensable for Golgi localization of Sial-EGFP. (A) Western blot demonstrates knockdown of GOLPH3L and GOLPH3 in HEK 293 cells expressing Sial-EGFP. Lysates from control siRNA–treated cells are loaded at different relative amounts to provide a standard curve for semiquantitative assessment of knockdown efficiency. GAPDH Western blot demonstrates approximately equal loading. (B) HEK 293 cells expressing Sial-EGFP (green) were fixed and stained for IF to GOLPH3 (magenta), GM130 to mark the Golgi (red), and DAPI to label the nucleus (blue). The Golgi is expanded compared with controls when GOLPH3L is knocked down, and the Golgi is condensed compared with controls when GOLPH3 is knocked down. However, in all cases Sial-EGFP remains tightly localized to the Golgi. Bar, 10 μm. Inset, a magnified view of the corresponding boxed region. Similar results with α-mannosidase II-EGFP and β-1,4-galactosyltransferase-EYFP are included in Supplemental Figures S4 and S5.

Article Snippet: Mammalian cell lines, including HEK 293 (AD-293 variant; Stratagene, Santa Clara, CA), NIH 3T3 fibroblasts, HeLa S3, LNCaP, MCF-7, Neuro2A, NRK, N-Tera 2, RAW macrophages, and U2-OS cells, were grown according to American Type Culture Collection (Manassas, VA) guidelines.

Techniques: Western Blot, Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

doi: 10.1091/mbc.E12-07-0525

Figure Lengend Snippet: GOLPH3L does not share GOLPH3’s tight interaction with MYO18A. (A) IP of endogenous proteins from HEK 293 cell lysates using antibodies for GOLPH3, GOLPH3L, or preimmune serum. Preimmune serum fails to immunoprecipitate GOLPH3, GOLPH3L, or MYO18A. GOLPH3 (but not GOLPH3L) is specifically immunoprecipitated using GOLPH3 antibody, and MYO18A coimmunoprecipitates with GOLPH3. GOLPH3L (but not GOLPH3) is specifically immunoprecipitated using GOLPH3L antibody; however, MYO18A does not appreciably coimmunoprecipitate with GOLPH3L. (B) IP using antibodies to GOLPH3 or GOLPH3L from control or GOLPH3 knockdown lysates from HEK 293 cells. In control lysates GOLPH3 (but not GOLPH3L) is specifically immunoprecipitated using GOLPH3 antibody, and MYO18A coimmunoprecipitates with GOLPH3. GOLPH3L (but not GOLPH3) is specifically immunoprecipitated using GOLPH3L antibody; however MYO18A does not coimmunoprecipitate with GOLPH3L. In GOLPH3-knockdown lysates GOLPH3 levels are greatly reduced. Despite reduced GOLPH3 levels, specific IP of GOLPH3L still does not appreciably coimmunoprecipitate MYO18A.

Article Snippet: Mammalian cell lines, including HEK 293 (AD-293 variant; Stratagene, Santa Clara, CA), NIH 3T3 fibroblasts, HeLa S3, LNCaP, MCF-7, Neuro2A, NRK, N-Tera 2, RAW macrophages, and U2-OS cells, were grown according to American Type Culture Collection (Manassas, VA) guidelines.

Techniques: Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

doi: 10.1091/mbc.E12-07-0525

Figure Lengend Snippet: Efficient secretion in HEK 293 cells is dependent on both GOLPH3 and GOLPH3L. In HEK 293 cells, a pulse-chase assay was used to measure total protein secretion as described in Materials and Methods . Graph shows mean secretion relative to control siRNA–treated cells with SEM and number of replicates (pooled from three independent experiments) as indicated. Control cells treated with brefeldin A, as well as cells with siRNA knockdown of GOLPH3, MYO18A, or GOLPH3L, all have significantly decreased total secretory flux compared with control. Statistical significance is indicated ( t test).

Article Snippet: Mammalian cell lines, including HEK 293 (AD-293 variant; Stratagene, Santa Clara, CA), NIH 3T3 fibroblasts, HeLa S3, LNCaP, MCF-7, Neuro2A, NRK, N-Tera 2, RAW macrophages, and U2-OS cells, were grown according to American Type Culture Collection (Manassas, VA) guidelines.

Techniques: Pulse Chase