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rilpl2 knockout mouse line  (Mutant Mouse Resource & Research Center)

 
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    Mutant Mouse Resource & Research Center rilpl2 knockout mouse line
    (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit <t>anti-RILPL2</t> (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.
    Rilpl2 Knockout Mouse Line, supplied by Mutant Mouse Resource & Research Center, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rilpl2+knockout+mouse+line/rilpl2+knockout+mouse+line/pmc07994366-212-0-16
    Average 90 stars, based on 1 article reviews
    rilpl2 knockout mouse line - by Bioz Stars, 2026-09
    90/100 stars

    Images

    1) Product Images from "LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade"

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202101050

    (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit anti-RILPL2 (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.
    Figure Legend Snippet: (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit anti-RILPL2 (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.

    Techniques Used: Staining, Transfection, Quantitation Assay, Expressing

    (A) RPE cells transfected with HA-RILP2 or GFP-RILPL2 as indicated and stained for endogenous MyoVa. Bars, 10 μm. (B) R1441C MEFs transfected with HA-RILPL2, ± MLi-2 LRRK2 inhibitor. Cells were serum starved for 16 h before fixation and staining with mouse anti-CEP164 or rabbit anti-HA. Bars, 10 μm. Boxes indicate the areas enlarged at the right. (C) Quantitation of pericentriolar fluorescence intensity in arbitrary units ±MLi-2. * P = 0.024. (D) HEK293T cells depleted of MyoVa as indicated and transfected with GFP-R1441G LRRK2 for 24 h. Cells were stained with rabbit anti-pRab10 antibody. (D, E) Quantitation of percent of cells with perinuclear, clustered Rab10 in control or MyoVa shRNA treated cells as in (D).
    Figure Legend Snippet: (A) RPE cells transfected with HA-RILP2 or GFP-RILPL2 as indicated and stained for endogenous MyoVa. Bars, 10 μm. (B) R1441C MEFs transfected with HA-RILPL2, ± MLi-2 LRRK2 inhibitor. Cells were serum starved for 16 h before fixation and staining with mouse anti-CEP164 or rabbit anti-HA. Bars, 10 μm. Boxes indicate the areas enlarged at the right. (C) Quantitation of pericentriolar fluorescence intensity in arbitrary units ±MLi-2. * P = 0.024. (D) HEK293T cells depleted of MyoVa as indicated and transfected with GFP-R1441G LRRK2 for 24 h. Cells were stained with rabbit anti-pRab10 antibody. (D, E) Quantitation of percent of cells with perinuclear, clustered Rab10 in control or MyoVa shRNA treated cells as in (D).

    Techniques Used: Transfection, Staining, Quantitation Assay, Fluorescence, shRNA

    (A) Localization of pRab10 and the centriole marker, Centrin 3. HeLa cells were transfected with R1441G Myc-LRRK2 and methanol fixed for staining with rabbit anti-pRab10 and mouse anti-Centrin 3. The boxed area is enlarged in the adjacent panels at right. Bar, 10 μM. (B) HEK293T cells were transfected with R1441G LRRK2, full-length MyoVa mCherry, and HA-RILPL2 for 16 h, treated ± 200 nM MLi-2 for 2 h and stained with mouse anti-HA and rabbit anti-phosphoRab10. Bar, 10 μm. (C) Quantitation of cells with perinuclear RILPL2 from the experiment in (B) from two independent experiments with >50 cells per condition. *0.027; **0.0026 t test. (B, D) HEK293T cells depleted of MyoVa were transfected with R1441G LRRK2 and HA-RILPL2 and stained for RILPL2 and pRab10 as in (B). (E) Immunoblot of HEK293T cells depleted of MyoVa using three shRNAs to create stable cell lines. Numbers at left in this and subsequent figures represent mass in kilodaltons. (F) Quantitation of perinuclear RILPL2 in cells with and without MyoVa. No significant difference was detected.
    Figure Legend Snippet: (A) Localization of pRab10 and the centriole marker, Centrin 3. HeLa cells were transfected with R1441G Myc-LRRK2 and methanol fixed for staining with rabbit anti-pRab10 and mouse anti-Centrin 3. The boxed area is enlarged in the adjacent panels at right. Bar, 10 μM. (B) HEK293T cells were transfected with R1441G LRRK2, full-length MyoVa mCherry, and HA-RILPL2 for 16 h, treated ± 200 nM MLi-2 for 2 h and stained with mouse anti-HA and rabbit anti-phosphoRab10. Bar, 10 μm. (C) Quantitation of cells with perinuclear RILPL2 from the experiment in (B) from two independent experiments with >50 cells per condition. *0.027; **0.0026 t test. (B, D) HEK293T cells depleted of MyoVa were transfected with R1441G LRRK2 and HA-RILPL2 and stained for RILPL2 and pRab10 as in (B). (E) Immunoblot of HEK293T cells depleted of MyoVa using three shRNAs to create stable cell lines. Numbers at left in this and subsequent figures represent mass in kilodaltons. (F) Quantitation of perinuclear RILPL2 in cells with and without MyoVa. No significant difference was detected.

    Techniques Used: Marker, Transfection, Staining, Quantitation Assay, Western Blot, Stable Transfection

    (A) HEK293T cells were co-transfected with Myc-LRRK2-R1441G and MyoVa-mCherry. After 18 h, cells were fixed and stained with mouse anti-Myc (blue) and rabbit anti-pRab10 (cyan). (B) Quantitation of colocalization by Mander’s method between MyoVa and pRab10. The fraction of MyoVa that colocalizes with pRab10 and vice-versa are quantified. Error bars indicate standard error of the mean of colocalization coefficient for >20 cells. (A, C) HEK293T cells depleted of Rab10 (KD) were co-transfected with Myc-LRRK2 R1441G and HA-RILPL2. (A) After 18 h, the cells were stained as in (A). (D) R1441G LRRK2 MEF cells, either control or depleted of RILPL2, transfected with globular tail domain-mCherry. Markers are indicated; cells were stained wisth mouse anti-CEP164 or rabbit anti-mCherry. (E) Quantitation of the experiment shown in (D); P -values were determined by t test: ***0.0009 and 0.0004. (F) qPCR quantitation of RILPL2 mRNA in cells treated with two different shRNAs as indicated.
    Figure Legend Snippet: (A) HEK293T cells were co-transfected with Myc-LRRK2-R1441G and MyoVa-mCherry. After 18 h, cells were fixed and stained with mouse anti-Myc (blue) and rabbit anti-pRab10 (cyan). (B) Quantitation of colocalization by Mander’s method between MyoVa and pRab10. The fraction of MyoVa that colocalizes with pRab10 and vice-versa are quantified. Error bars indicate standard error of the mean of colocalization coefficient for >20 cells. (A, C) HEK293T cells depleted of Rab10 (KD) were co-transfected with Myc-LRRK2 R1441G and HA-RILPL2. (A) After 18 h, the cells were stained as in (A). (D) R1441G LRRK2 MEF cells, either control or depleted of RILPL2, transfected with globular tail domain-mCherry. Markers are indicated; cells were stained wisth mouse anti-CEP164 or rabbit anti-mCherry. (E) Quantitation of the experiment shown in (D); P -values were determined by t test: ***0.0009 and 0.0004. (F) qPCR quantitation of RILPL2 mRNA in cells treated with two different shRNAs as indicated.

    Techniques Used: Transfection, Staining, Quantitation Assay

    (A) MyoVa or MyoVa ΔD-mCherry and GFP-LRRK2-R1441G were co-transfected into HEK293T cells; 24 h post transfection, cells were incubated with 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and 400 μg extract immunoprecipitated with anti-RFP antibodies on protein G beads. Samples (50% of IP and 15% of input for all IPs in all panels) were immunoblotted using anti-GFP, anti-RFP, anti-tubulin, anti-Rab10 and anti-pRab10 antibodies. (A, B) Quantitation of the relative amount of Rab10 (left) or pRab10 (right) co-immunoprecipitated by MyoVa and MyoVa ΔD in (A). Error bars indicate standard error of the mean from two gels per co-IP. Significance was determined by t test; *** P = 0.0003; ns, not significant with P = 0.295. (C) MyoVa globular tail domain (GTD)-mCherry and GFP-LRRK2 R1441G were co-transfected into HEK293T cells. After 24 h, cells were incubated ± 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and immunoprecipitated with anti-RFP antibodies on protein G beads. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D) RILPL2-GFP, MyoVa GTD-mCherry, and Myc-LRRK2 R1441G were co-transfected into HEK293T cells; 24 h post-transfection, cells were incubated ± 200 nM MLi-2 for 4 h. Extracts in lysis buffer were immunoprecipitated with GFP-binding protein Sepharose. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D, E) Quantitation of the relative amount of total MyoVa GTD (left) and Rab10 (right) co-immunoprecipitated by RILPL2-GFP under the conditions indicated below (E). Significance was determined by t test; * P = 0.021 and 0.035 (left) and P = 0.014 and 0.024 (right).
    Figure Legend Snippet: (A) MyoVa or MyoVa ΔD-mCherry and GFP-LRRK2-R1441G were co-transfected into HEK293T cells; 24 h post transfection, cells were incubated with 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and 400 μg extract immunoprecipitated with anti-RFP antibodies on protein G beads. Samples (50% of IP and 15% of input for all IPs in all panels) were immunoblotted using anti-GFP, anti-RFP, anti-tubulin, anti-Rab10 and anti-pRab10 antibodies. (A, B) Quantitation of the relative amount of Rab10 (left) or pRab10 (right) co-immunoprecipitated by MyoVa and MyoVa ΔD in (A). Error bars indicate standard error of the mean from two gels per co-IP. Significance was determined by t test; *** P = 0.0003; ns, not significant with P = 0.295. (C) MyoVa globular tail domain (GTD)-mCherry and GFP-LRRK2 R1441G were co-transfected into HEK293T cells. After 24 h, cells were incubated ± 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and immunoprecipitated with anti-RFP antibodies on protein G beads. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D) RILPL2-GFP, MyoVa GTD-mCherry, and Myc-LRRK2 R1441G were co-transfected into HEK293T cells; 24 h post-transfection, cells were incubated ± 200 nM MLi-2 for 4 h. Extracts in lysis buffer were immunoprecipitated with GFP-binding protein Sepharose. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D, E) Quantitation of the relative amount of total MyoVa GTD (left) and Rab10 (right) co-immunoprecipitated by RILPL2-GFP under the conditions indicated below (E). Significance was determined by t test; * P = 0.021 and 0.035 (left) and P = 0.014 and 0.024 (right).

    Techniques Used: Transfection, Incubation, Lysis, Immunoprecipitation, Quantitation Assay, Co-Immunoprecipitation Assay, Binding Assay

    (A) hTERT-RPE cells were transfected with HA-RILPL2 or mock transfected with lipofectamine and 24 h later, serum-starved overnight to initiate ciliogenesis. Cells were fixed with ice cold −20°C methanol for 2 min and co-stained with rabbit anti-HA (green) or mouse anti-Arl13b (red). Arrowhead indicates the likely location of a RILPL2-positive centriole. Dotted lines indicate cell outlines. Bar, 10 μm. (B) Quantitation of cells with cilia ± HA-RILPL2 transfection. Error bars represent SEM from two experiments, each with >25 cells per condition. Significance was determined by t test; * P = 0.0164. (C) WT, RILPL1, and RILPL2 knockout A549 cells were plated at 80% confluency. After 24 h, cells were serum starved by transfer into 2% FBS-containing medium for 48 h. Cells were fixed and stained with mouse anti-Arl13b antibody for quantitation of cells with cilia. Error bars represent SEM from two experiments with >100 cells per condition in each experiment. #1 and #2 indicate two different A549 RILPL2 knockout clonal cell lines. (D) Quantitation of cells with two CP110 dots indicating capped centrioles in: (left) hTERT-RPE cells ± HA-RILPL2 for 24 h; (right) hTERT-RPE cells ± MyoVa knock-down. Cells were serum starved for 2 h, fixed with −20°C methanol for 2 min and co-stained with anti-HA, anti-CP110 or anti-CEP164/CP110 antibodies. Error bars represent SEM from two experiments with >25 cells per condition in each experiment. Significance was determined by t test; ** P = 0.0067 and 0.0045. (B, E) RPE cells expressing HA-RILPL2 as in panel (B) were scored for TTBK2 at the centriole after serum starvation as in . Shown are data from two independent experiments; >25 cells per experiment. No significant difference was detected. (B, F) RPE cells expressing HA-RILPL2 as in panel (B) were scored for endogenous MyoVa at the centriole; shown are data from two independent experiments; >50 cells per experiment for each condition. **** P < 0.0001 by t test.
    Figure Legend Snippet: (A) hTERT-RPE cells were transfected with HA-RILPL2 or mock transfected with lipofectamine and 24 h later, serum-starved overnight to initiate ciliogenesis. Cells were fixed with ice cold −20°C methanol for 2 min and co-stained with rabbit anti-HA (green) or mouse anti-Arl13b (red). Arrowhead indicates the likely location of a RILPL2-positive centriole. Dotted lines indicate cell outlines. Bar, 10 μm. (B) Quantitation of cells with cilia ± HA-RILPL2 transfection. Error bars represent SEM from two experiments, each with >25 cells per condition. Significance was determined by t test; * P = 0.0164. (C) WT, RILPL1, and RILPL2 knockout A549 cells were plated at 80% confluency. After 24 h, cells were serum starved by transfer into 2% FBS-containing medium for 48 h. Cells were fixed and stained with mouse anti-Arl13b antibody for quantitation of cells with cilia. Error bars represent SEM from two experiments with >100 cells per condition in each experiment. #1 and #2 indicate two different A549 RILPL2 knockout clonal cell lines. (D) Quantitation of cells with two CP110 dots indicating capped centrioles in: (left) hTERT-RPE cells ± HA-RILPL2 for 24 h; (right) hTERT-RPE cells ± MyoVa knock-down. Cells were serum starved for 2 h, fixed with −20°C methanol for 2 min and co-stained with anti-HA, anti-CP110 or anti-CEP164/CP110 antibodies. Error bars represent SEM from two experiments with >25 cells per condition in each experiment. Significance was determined by t test; ** P = 0.0067 and 0.0045. (B, E) RPE cells expressing HA-RILPL2 as in panel (B) were scored for TTBK2 at the centriole after serum starvation as in . Shown are data from two independent experiments; >25 cells per experiment. No significant difference was detected. (B, F) RPE cells expressing HA-RILPL2 as in panel (B) were scored for endogenous MyoVa at the centriole; shown are data from two independent experiments; >50 cells per experiment for each condition. **** P < 0.0001 by t test.

    Techniques Used: Transfection, Staining, Quantitation Assay, Knock-Out, Expressing

    (A) Top row, RPE cells transfected with HA-RILPL2 (red) and stained for rabbit anti-CP110 (green). Cells were serum-starved for 2 h before fixation and staining. Inset 1, RILPL2 transfected cell; inset 2 is a non-RILPL2 transfected cell. Second row, MyoVa-depleted RPE cells; shown is CP110 and CEP164 staining, with the boxed area enlarged at right. (B) TTBK2 and centriole marker, rootletin localization in RPE cells expressing HA-RILPL2 or HA alone as indicated. Boxed regions in the first column are enlarged at right. (C) As in (B), monitoring MyoVa and rootletin localization. Shown are two cells, with and without RILPL2 expression.
    Figure Legend Snippet: (A) Top row, RPE cells transfected with HA-RILPL2 (red) and stained for rabbit anti-CP110 (green). Cells were serum-starved for 2 h before fixation and staining. Inset 1, RILPL2 transfected cell; inset 2 is a non-RILPL2 transfected cell. Second row, MyoVa-depleted RPE cells; shown is CP110 and CEP164 staining, with the boxed area enlarged at right. (B) TTBK2 and centriole marker, rootletin localization in RPE cells expressing HA-RILPL2 or HA alone as indicated. Boxed regions in the first column are enlarged at right. (C) As in (B), monitoring MyoVa and rootletin localization. Shown are two cells, with and without RILPL2 expression.

    Techniques Used: Transfection, Staining, Marker, Expressing

    Related Articles

    Knock-Out:

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade
    Article Snippet: .. RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD). .. Rilpl2 tm1a(KOMP)Wtsi mice were initially bred with Taconic Total Body Cre mice expressing Cre recombinase (Model 12524) to produce the desired deletion.



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    rilpl2 knockout mouse line  (Mutant Mouse Resource & Research Center)
    90
    Mutant Mouse Resource & Research Center rilpl2 knockout mouse line
    (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit <t>anti-RILPL2</t> (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.
    Rilpl2 Knockout Mouse Line, supplied by Mutant Mouse Resource & Research Center, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rilpl2+knockout+mouse+line/rilpl2+knockout+mouse+line/pmc07994366-212-0-16
    Average 90 stars, based on 1 article reviews
    rilpl2 knockout mouse line - by Bioz Stars, 2026-09
    90/100 stars
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    (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit anti-RILPL2 (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) Primary, wild type rat astrocytes were fixed with 3% PFA and stained for cilia using mouse anti-Arl13b (green) and rabbit anti-RILPL2 (red). Box indicates the region enlarged in the image shown at right. Bar, 10 μm. Arrows indicate the base of the cilium. (B) hTERT-RPE cells were transfected with HA-RILPL2 at 80% confluency and 24 h later either medium changed (+FBS) or serum starved (−FBS) for 2 h. Cells were fixed with −20°C methanol for 2 min and stained with rabbit or mouse anti-HA to detect RILPL2 (red) and mouse anti-CEP164, or rabbit anti-CP110 (green). Arrows indicate the location of the centriolar region. Bar, 2.5 μm. (C) Quantitation of percent of cells that show centriolar localized RILPL2 ± 2 h serum starvation. Significance was determined by t test; * P = 0.03. Error bars represent standard error of the mean from two independent experiments with >50 cells per condition. (D) hTERT-RPE cells expressing mKO2-PACT (magenta) were transfected with GFP-RILPL2 (green). Cells were serum starved and 15 min later imaged by capturing Z-stacks every 6 min for the next 8 h. Yellow arrowheads indicate the location of the centrosome as marked by mKO2-PACT. Upper panel shows GFP-RILPL2 in grayscale. Scale bar, 5 μm. (E) Times at which GFP-RILPL2 appeared at the mKO2-PACT structure. Error bar represents standard error of the mean.

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Staining, Transfection, Quantitation Assay, Expressing

    (A) RPE cells transfected with HA-RILP2 or GFP-RILPL2 as indicated and stained for endogenous MyoVa. Bars, 10 μm. (B) R1441C MEFs transfected with HA-RILPL2, ± MLi-2 LRRK2 inhibitor. Cells were serum starved for 16 h before fixation and staining with mouse anti-CEP164 or rabbit anti-HA. Bars, 10 μm. Boxes indicate the areas enlarged at the right. (C) Quantitation of pericentriolar fluorescence intensity in arbitrary units ±MLi-2. * P = 0.024. (D) HEK293T cells depleted of MyoVa as indicated and transfected with GFP-R1441G LRRK2 for 24 h. Cells were stained with rabbit anti-pRab10 antibody. (D, E) Quantitation of percent of cells with perinuclear, clustered Rab10 in control or MyoVa shRNA treated cells as in (D).

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) RPE cells transfected with HA-RILP2 or GFP-RILPL2 as indicated and stained for endogenous MyoVa. Bars, 10 μm. (B) R1441C MEFs transfected with HA-RILPL2, ± MLi-2 LRRK2 inhibitor. Cells were serum starved for 16 h before fixation and staining with mouse anti-CEP164 or rabbit anti-HA. Bars, 10 μm. Boxes indicate the areas enlarged at the right. (C) Quantitation of pericentriolar fluorescence intensity in arbitrary units ±MLi-2. * P = 0.024. (D) HEK293T cells depleted of MyoVa as indicated and transfected with GFP-R1441G LRRK2 for 24 h. Cells were stained with rabbit anti-pRab10 antibody. (D, E) Quantitation of percent of cells with perinuclear, clustered Rab10 in control or MyoVa shRNA treated cells as in (D).

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Transfection, Staining, Quantitation Assay, Fluorescence, shRNA

    (A) Localization of pRab10 and the centriole marker, Centrin 3. HeLa cells were transfected with R1441G Myc-LRRK2 and methanol fixed for staining with rabbit anti-pRab10 and mouse anti-Centrin 3. The boxed area is enlarged in the adjacent panels at right. Bar, 10 μM. (B) HEK293T cells were transfected with R1441G LRRK2, full-length MyoVa mCherry, and HA-RILPL2 for 16 h, treated ± 200 nM MLi-2 for 2 h and stained with mouse anti-HA and rabbit anti-phosphoRab10. Bar, 10 μm. (C) Quantitation of cells with perinuclear RILPL2 from the experiment in (B) from two independent experiments with >50 cells per condition. *0.027; **0.0026 t test. (B, D) HEK293T cells depleted of MyoVa were transfected with R1441G LRRK2 and HA-RILPL2 and stained for RILPL2 and pRab10 as in (B). (E) Immunoblot of HEK293T cells depleted of MyoVa using three shRNAs to create stable cell lines. Numbers at left in this and subsequent figures represent mass in kilodaltons. (F) Quantitation of perinuclear RILPL2 in cells with and without MyoVa. No significant difference was detected.

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) Localization of pRab10 and the centriole marker, Centrin 3. HeLa cells were transfected with R1441G Myc-LRRK2 and methanol fixed for staining with rabbit anti-pRab10 and mouse anti-Centrin 3. The boxed area is enlarged in the adjacent panels at right. Bar, 10 μM. (B) HEK293T cells were transfected with R1441G LRRK2, full-length MyoVa mCherry, and HA-RILPL2 for 16 h, treated ± 200 nM MLi-2 for 2 h and stained with mouse anti-HA and rabbit anti-phosphoRab10. Bar, 10 μm. (C) Quantitation of cells with perinuclear RILPL2 from the experiment in (B) from two independent experiments with >50 cells per condition. *0.027; **0.0026 t test. (B, D) HEK293T cells depleted of MyoVa were transfected with R1441G LRRK2 and HA-RILPL2 and stained for RILPL2 and pRab10 as in (B). (E) Immunoblot of HEK293T cells depleted of MyoVa using three shRNAs to create stable cell lines. Numbers at left in this and subsequent figures represent mass in kilodaltons. (F) Quantitation of perinuclear RILPL2 in cells with and without MyoVa. No significant difference was detected.

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Marker, Transfection, Staining, Quantitation Assay, Western Blot, Stable Transfection

    (A) HEK293T cells were co-transfected with Myc-LRRK2-R1441G and MyoVa-mCherry. After 18 h, cells were fixed and stained with mouse anti-Myc (blue) and rabbit anti-pRab10 (cyan). (B) Quantitation of colocalization by Mander’s method between MyoVa and pRab10. The fraction of MyoVa that colocalizes with pRab10 and vice-versa are quantified. Error bars indicate standard error of the mean of colocalization coefficient for >20 cells. (A, C) HEK293T cells depleted of Rab10 (KD) were co-transfected with Myc-LRRK2 R1441G and HA-RILPL2. (A) After 18 h, the cells were stained as in (A). (D) R1441G LRRK2 MEF cells, either control or depleted of RILPL2, transfected with globular tail domain-mCherry. Markers are indicated; cells were stained wisth mouse anti-CEP164 or rabbit anti-mCherry. (E) Quantitation of the experiment shown in (D); P -values were determined by t test: ***0.0009 and 0.0004. (F) qPCR quantitation of RILPL2 mRNA in cells treated with two different shRNAs as indicated.

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) HEK293T cells were co-transfected with Myc-LRRK2-R1441G and MyoVa-mCherry. After 18 h, cells were fixed and stained with mouse anti-Myc (blue) and rabbit anti-pRab10 (cyan). (B) Quantitation of colocalization by Mander’s method between MyoVa and pRab10. The fraction of MyoVa that colocalizes with pRab10 and vice-versa are quantified. Error bars indicate standard error of the mean of colocalization coefficient for >20 cells. (A, C) HEK293T cells depleted of Rab10 (KD) were co-transfected with Myc-LRRK2 R1441G and HA-RILPL2. (A) After 18 h, the cells were stained as in (A). (D) R1441G LRRK2 MEF cells, either control or depleted of RILPL2, transfected with globular tail domain-mCherry. Markers are indicated; cells were stained wisth mouse anti-CEP164 or rabbit anti-mCherry. (E) Quantitation of the experiment shown in (D); P -values were determined by t test: ***0.0009 and 0.0004. (F) qPCR quantitation of RILPL2 mRNA in cells treated with two different shRNAs as indicated.

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Transfection, Staining, Quantitation Assay

    (A) MyoVa or MyoVa ΔD-mCherry and GFP-LRRK2-R1441G were co-transfected into HEK293T cells; 24 h post transfection, cells were incubated with 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and 400 μg extract immunoprecipitated with anti-RFP antibodies on protein G beads. Samples (50% of IP and 15% of input for all IPs in all panels) were immunoblotted using anti-GFP, anti-RFP, anti-tubulin, anti-Rab10 and anti-pRab10 antibodies. (A, B) Quantitation of the relative amount of Rab10 (left) or pRab10 (right) co-immunoprecipitated by MyoVa and MyoVa ΔD in (A). Error bars indicate standard error of the mean from two gels per co-IP. Significance was determined by t test; *** P = 0.0003; ns, not significant with P = 0.295. (C) MyoVa globular tail domain (GTD)-mCherry and GFP-LRRK2 R1441G were co-transfected into HEK293T cells. After 24 h, cells were incubated ± 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and immunoprecipitated with anti-RFP antibodies on protein G beads. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D) RILPL2-GFP, MyoVa GTD-mCherry, and Myc-LRRK2 R1441G were co-transfected into HEK293T cells; 24 h post-transfection, cells were incubated ± 200 nM MLi-2 for 4 h. Extracts in lysis buffer were immunoprecipitated with GFP-binding protein Sepharose. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D, E) Quantitation of the relative amount of total MyoVa GTD (left) and Rab10 (right) co-immunoprecipitated by RILPL2-GFP under the conditions indicated below (E). Significance was determined by t test; * P = 0.021 and 0.035 (left) and P = 0.014 and 0.024 (right).

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) MyoVa or MyoVa ΔD-mCherry and GFP-LRRK2-R1441G were co-transfected into HEK293T cells; 24 h post transfection, cells were incubated with 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and 400 μg extract immunoprecipitated with anti-RFP antibodies on protein G beads. Samples (50% of IP and 15% of input for all IPs in all panels) were immunoblotted using anti-GFP, anti-RFP, anti-tubulin, anti-Rab10 and anti-pRab10 antibodies. (A, B) Quantitation of the relative amount of Rab10 (left) or pRab10 (right) co-immunoprecipitated by MyoVa and MyoVa ΔD in (A). Error bars indicate standard error of the mean from two gels per co-IP. Significance was determined by t test; *** P = 0.0003; ns, not significant with P = 0.295. (C) MyoVa globular tail domain (GTD)-mCherry and GFP-LRRK2 R1441G were co-transfected into HEK293T cells. After 24 h, cells were incubated ± 200 nM MLi-2 for 4 h. Cells were lysed in lysis buffer and immunoprecipitated with anti-RFP antibodies on protein G beads. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D) RILPL2-GFP, MyoVa GTD-mCherry, and Myc-LRRK2 R1441G were co-transfected into HEK293T cells; 24 h post-transfection, cells were incubated ± 200 nM MLi-2 for 4 h. Extracts in lysis buffer were immunoprecipitated with GFP-binding protein Sepharose. Samples were immunoblotted with anti-GFP, anti-RFP, anti-Rab10 and anti-pRab10 antibodies. (D, E) Quantitation of the relative amount of total MyoVa GTD (left) and Rab10 (right) co-immunoprecipitated by RILPL2-GFP under the conditions indicated below (E). Significance was determined by t test; * P = 0.021 and 0.035 (left) and P = 0.014 and 0.024 (right).

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Transfection, Incubation, Lysis, Immunoprecipitation, Quantitation Assay, Co-Immunoprecipitation Assay, Binding Assay

    (A) hTERT-RPE cells were transfected with HA-RILPL2 or mock transfected with lipofectamine and 24 h later, serum-starved overnight to initiate ciliogenesis. Cells were fixed with ice cold −20°C methanol for 2 min and co-stained with rabbit anti-HA (green) or mouse anti-Arl13b (red). Arrowhead indicates the likely location of a RILPL2-positive centriole. Dotted lines indicate cell outlines. Bar, 10 μm. (B) Quantitation of cells with cilia ± HA-RILPL2 transfection. Error bars represent SEM from two experiments, each with >25 cells per condition. Significance was determined by t test; * P = 0.0164. (C) WT, RILPL1, and RILPL2 knockout A549 cells were plated at 80% confluency. After 24 h, cells were serum starved by transfer into 2% FBS-containing medium for 48 h. Cells were fixed and stained with mouse anti-Arl13b antibody for quantitation of cells with cilia. Error bars represent SEM from two experiments with >100 cells per condition in each experiment. #1 and #2 indicate two different A549 RILPL2 knockout clonal cell lines. (D) Quantitation of cells with two CP110 dots indicating capped centrioles in: (left) hTERT-RPE cells ± HA-RILPL2 for 24 h; (right) hTERT-RPE cells ± MyoVa knock-down. Cells were serum starved for 2 h, fixed with −20°C methanol for 2 min and co-stained with anti-HA, anti-CP110 or anti-CEP164/CP110 antibodies. Error bars represent SEM from two experiments with >25 cells per condition in each experiment. Significance was determined by t test; ** P = 0.0067 and 0.0045. (B, E) RPE cells expressing HA-RILPL2 as in panel (B) were scored for TTBK2 at the centriole after serum starvation as in . Shown are data from two independent experiments; >25 cells per experiment. No significant difference was detected. (B, F) RPE cells expressing HA-RILPL2 as in panel (B) were scored for endogenous MyoVa at the centriole; shown are data from two independent experiments; >50 cells per experiment for each condition. **** P < 0.0001 by t test.

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) hTERT-RPE cells were transfected with HA-RILPL2 or mock transfected with lipofectamine and 24 h later, serum-starved overnight to initiate ciliogenesis. Cells were fixed with ice cold −20°C methanol for 2 min and co-stained with rabbit anti-HA (green) or mouse anti-Arl13b (red). Arrowhead indicates the likely location of a RILPL2-positive centriole. Dotted lines indicate cell outlines. Bar, 10 μm. (B) Quantitation of cells with cilia ± HA-RILPL2 transfection. Error bars represent SEM from two experiments, each with >25 cells per condition. Significance was determined by t test; * P = 0.0164. (C) WT, RILPL1, and RILPL2 knockout A549 cells were plated at 80% confluency. After 24 h, cells were serum starved by transfer into 2% FBS-containing medium for 48 h. Cells were fixed and stained with mouse anti-Arl13b antibody for quantitation of cells with cilia. Error bars represent SEM from two experiments with >100 cells per condition in each experiment. #1 and #2 indicate two different A549 RILPL2 knockout clonal cell lines. (D) Quantitation of cells with two CP110 dots indicating capped centrioles in: (left) hTERT-RPE cells ± HA-RILPL2 for 24 h; (right) hTERT-RPE cells ± MyoVa knock-down. Cells were serum starved for 2 h, fixed with −20°C methanol for 2 min and co-stained with anti-HA, anti-CP110 or anti-CEP164/CP110 antibodies. Error bars represent SEM from two experiments with >25 cells per condition in each experiment. Significance was determined by t test; ** P = 0.0067 and 0.0045. (B, E) RPE cells expressing HA-RILPL2 as in panel (B) were scored for TTBK2 at the centriole after serum starvation as in . Shown are data from two independent experiments; >25 cells per experiment. No significant difference was detected. (B, F) RPE cells expressing HA-RILPL2 as in panel (B) were scored for endogenous MyoVa at the centriole; shown are data from two independent experiments; >50 cells per experiment for each condition. **** P < 0.0001 by t test.

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Transfection, Staining, Quantitation Assay, Knock-Out, Expressing

    (A) Top row, RPE cells transfected with HA-RILPL2 (red) and stained for rabbit anti-CP110 (green). Cells were serum-starved for 2 h before fixation and staining. Inset 1, RILPL2 transfected cell; inset 2 is a non-RILPL2 transfected cell. Second row, MyoVa-depleted RPE cells; shown is CP110 and CEP164 staining, with the boxed area enlarged at right. (B) TTBK2 and centriole marker, rootletin localization in RPE cells expressing HA-RILPL2 or HA alone as indicated. Boxed regions in the first column are enlarged at right. (C) As in (B), monitoring MyoVa and rootletin localization. Shown are two cells, with and without RILPL2 expression.

    Journal: Life Science Alliance

    Article Title: LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

    doi: 10.26508/lsa.202101050

    Figure Lengend Snippet: (A) Top row, RPE cells transfected with HA-RILPL2 (red) and stained for rabbit anti-CP110 (green). Cells were serum-starved for 2 h before fixation and staining. Inset 1, RILPL2 transfected cell; inset 2 is a non-RILPL2 transfected cell. Second row, MyoVa-depleted RPE cells; shown is CP110 and CEP164 staining, with the boxed area enlarged at right. (B) TTBK2 and centriole marker, rootletin localization in RPE cells expressing HA-RILPL2 or HA alone as indicated. Boxed regions in the first column are enlarged at right. (C) As in (B), monitoring MyoVa and rootletin localization. Shown are two cells, with and without RILPL2 expression.

    Article Snippet: RILPL2 knockout mouse line was originally acquired from The KOMP Repository and is now available from MMRRC.org (Stock number: 049453-UCD).

    Techniques: Transfection, Staining, Marker, Expressing