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rab3d (Proteintech)

Name: rab3d
Brand: Proteintech
Rating: 93 (20 reviews)

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Proteintech rab3d

Decreased <t>Rab3D</t> and α-SMA expression in the LG of MFS mice. ( A ) Immunofluorescence staining showed strong Rab3D expression in the LGs of control mice, while 3-month-old MFS mice displayed a slight reduction in Rab3D fluorescence, with a more pronounced decrease in 10-month-old FBN1 mutants. Blue : DAPI. Scale bar : 20 µm. ( B ) Quantification of mean fluorescence intensity of Rab3D staining ( n = 5 mice/group). ( C ) Three-month-old control mice showed strong α-SMA expression in the LGs, while FBN1 mutants displayed slightly reduced fluorescence. Furthermore, α-SMA fluorescence was markedly lower in 10-month-old MFS mice. Blue : DAPI. Scale bar : 50 µm. ( D ) Quantitative histogram of α-SMA staining data ( n = 5 mice/group).

Rab3d, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 20 article reviews

rab3d - by Bioz Stars, 2026-02

93/100 stars

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1) Product Images from "Degradation of Elastic Fiber Triggers Lacrimal Gland Dysfunction in Marfan Syndrome Mice"

Article Title: Degradation of Elastic Fiber Triggers Lacrimal Gland Dysfunction in Marfan Syndrome Mice

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.66.15.63

Decreased Rab3D and α-SMA expression in the LG of MFS mice. ( A ) Immunofluorescence staining showed strong Rab3D expression in the LGs of control mice, while 3-month-old MFS mice displayed a slight reduction in Rab3D fluorescence, with a more pronounced decrease in 10-month-old FBN1 mutants. Blue : DAPI. Scale bar : 20 µm. ( B ) Quantification of mean fluorescence intensity of Rab3D staining ( n = 5 mice/group). ( C ) Three-month-old control mice showed strong α-SMA expression in the LGs, while FBN1 mutants displayed slightly reduced fluorescence. Furthermore, α-SMA fluorescence was markedly lower in 10-month-old MFS mice. Blue : DAPI. Scale bar : 50 µm. ( D ) Quantitative histogram of α-SMA staining data ( n = 5 mice/group).

Figure Legend Snippet: Decreased Rab3D and α-SMA expression in the LG of MFS mice. ( A ) Immunofluorescence staining showed strong Rab3D expression in the LGs of control mice, while 3-month-old MFS mice displayed a slight reduction in Rab3D fluorescence, with a more pronounced decrease in 10-month-old FBN1 mutants. Blue : DAPI. Scale bar : 20 µm. ( B ) Quantification of mean fluorescence intensity of Rab3D staining ( n = 5 mice/group). ( C ) Three-month-old control mice showed strong α-SMA expression in the LGs, while FBN1 mutants displayed slightly reduced fluorescence. Furthermore, α-SMA fluorescence was markedly lower in 10-month-old MFS mice. Blue : DAPI. Scale bar : 50 µm. ( D ) Quantitative histogram of α-SMA staining data ( n = 5 mice/group).

Techniques Used: Expressing, Immunofluorescence, Staining, Control, Fluorescence

Image Search Results

Journal: Investigative Ophthalmology & Visual Science

Article Title: Degradation of Elastic Fiber Triggers Lacrimal Gland Dysfunction in Marfan Syndrome Mice

doi: 10.1167/iovs.66.15.63

Figure Lengend Snippet: Decreased Rab3D and α-SMA expression in the LG of MFS mice. ( A ) Immunofluorescence staining showed strong Rab3D expression in the LGs of control mice, while 3-month-old MFS mice displayed a slight reduction in Rab3D fluorescence, with a more pronounced decrease in 10-month-old FBN1 mutants. Blue : DAPI. Scale bar : 20 µm. ( B ) Quantification of mean fluorescence intensity of Rab3D staining ( n = 5 mice/group). ( C ) Three-month-old control mice showed strong α-SMA expression in the LGs, while FBN1 mutants displayed slightly reduced fluorescence. Furthermore, α-SMA fluorescence was markedly lower in 10-month-old MFS mice. Blue : DAPI. Scale bar : 50 µm. ( D ) Quantitative histogram of α-SMA staining data ( n = 5 mice/group).

Article Snippet: Other primary antibodies used in this study include COL1A1 (α1 type 1 collagen) (72026T; Cell Signaling Technology, Danvers, MA, USA), CD45 (ab10558; Abcam), MMP2 (ab235167; Abcam), MMP9 (ab38898; Abcam), Rab3D (12320-1-AP; Proteintech, Rosemont, IL, USA), α<@150>smooth muscle actin (α-SMA; ab5694; Abcam), TGF-β1 (A25313; ABclonal, Woburn, MA, USA), Smad2/3 (ab202445; Abcam), Phospho-SMAD2/3 (ab254407; Abcam), CCL2/MCP1 (ab7202; Abcam), SPP1/osteopontin (ab218237; Abcam), integrin-β1/ITGB1 (ab183666; Abcam), Alexa-Fluor 488 goat anti-rabbit secondary antibody (111-545-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Alexa-Fluor 555 goat anti-rabbit secondary antibody (111-565-144; Jackson ImmunoResearch Laboratories), horseradish peroxidase (HRP)–conjugated secondary antibody (7074S; Cell Signaling Technology), and GAPDH (2118; Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence, Staining, Control, Fluorescence

A-B. Western blot (WB) analysis of ZDHHC20, Short-S (42 KDa) and 20L (∼49, 62 and 69 KDa), GAPDH (loading control) from lysates of: A. a panel of cancer cell lines and infected Calu-3 cells (SARS-CoV-2, MOI 0.1, 24 h) as comparison; B. primary human airway epithelia from individuals with different ages or infected with SARS-CoV-2 MOI□=□0.1, 48□h. C. Overview of the ZDHHC20 locus (first intron, exon, and 5’UTR - version hg19), Tracks indicate: in-frame transcription start codons (ATGs); transcript (GENCODE-V4 0lift37); histone modifications ChIP-seq signals with minimum and maximum data points in the current view (ENCODE/Broad) - H3K9me3, black, repression; enhancer, H3K4me1 green; activation, H3K7Ac, purple; or promoter, H3K4me3, pink; SETDB1/KAP1, ATF2, SP1 and FOXA1 ChIP-seq peaks (ENCODE) D . WB, as (A), also showing total and phospho-ATF2 in HeLa cells siControl (siCtrl) or siATF2 left untreated or treated with 10□ng/ml of proaerolysin (toxin) for 1hr at 37□°C, washed, and incubated at 37□°C for 8 h. Correspondent quantification of 20L bands are mean□±□SEM, each dot represents one independent experiment, n□=□10; P values obtained by two-way ANOVA, Sidak’s correction. E. WB as (A) in U2OS control or expressing ATF2 (WT, 6D or 6A phospho-mutants), and FOXA1 and SP1. Results are mean□±□SEM; each dot represents one independent experiment, n□=□6 (Ctrl) or n=3; P values compare to Ctrl, obtained by two-way ANOVA, Dunnett’s correction. F . Cell viability, monitored by ATP detection (Promega Glo - see materials and methods), in cells treated as (E). Values normalized to Untreated siCtrl, set as 100%. Results are mean ±□SD of one of three representative assays (n=34) replicates. P values, compared to siCtrl were obtained by two-way ANOVA, Dunnett’s correction. G. KAP1-KZFPs recruit histone modifiers, ( e.g. SETDB1) inducing H3K9me3-mediated chromatin repression (top), often involving removal of active marks at enhancers (H3K4me1), activation sites (H3K27Ac), and promoters (H3K4me3) (bottom) (adapted from ). H. As (D), in U2OS siCtrl, siSETDB1 or siKAP1-depleted for 72 h; Results are mean□±□SEM; each dot represents one independent experiment of n□=□6; P values comparing to Ctrl obtained by two-way ANOVA, Dunnett’s correction. I. As (H), with siSETDB1 cells complemented with WT or catalytically inactive SETDB1 (Mut) for 24 h. J . As (A), in HAP-1 wild-type or knockout for KAP1. K. mRNA quantification probing different locations in Z DHHC20 transcripts (coding region: C1, C2; 5’ UTR: 1– 5 cover increasing lengths of 5’ ends.

Journal: bioRxiv

Article Title: Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

doi: 10.1101/2025.02.25.640160

Figure Lengend Snippet: A-B. Western blot (WB) analysis of ZDHHC20, Short-S (42 KDa) and 20L (∼49, 62 and 69 KDa), GAPDH (loading control) from lysates of: A. a panel of cancer cell lines and infected Calu-3 cells (SARS-CoV-2, MOI 0.1, 24 h) as comparison; B. primary human airway epithelia from individuals with different ages or infected with SARS-CoV-2 MOI□=□0.1, 48□h. C. Overview of the ZDHHC20 locus (first intron, exon, and 5’UTR - version hg19), Tracks indicate: in-frame transcription start codons (ATGs); transcript (GENCODE-V4 0lift37); histone modifications ChIP-seq signals with minimum and maximum data points in the current view (ENCODE/Broad) - H3K9me3, black, repression; enhancer, H3K4me1 green; activation, H3K7Ac, purple; or promoter, H3K4me3, pink; SETDB1/KAP1, ATF2, SP1 and FOXA1 ChIP-seq peaks (ENCODE) D . WB, as (A), also showing total and phospho-ATF2 in HeLa cells siControl (siCtrl) or siATF2 left untreated or treated with 10□ng/ml of proaerolysin (toxin) for 1hr at 37□°C, washed, and incubated at 37□°C for 8 h. Correspondent quantification of 20L bands are mean□±□SEM, each dot represents one independent experiment, n□=□10; P values obtained by two-way ANOVA, Sidak’s correction. E. WB as (A) in U2OS control or expressing ATF2 (WT, 6D or 6A phospho-mutants), and FOXA1 and SP1. Results are mean□±□SEM; each dot represents one independent experiment, n□=□6 (Ctrl) or n=3; P values compare to Ctrl, obtained by two-way ANOVA, Dunnett’s correction. F . Cell viability, monitored by ATP detection (Promega Glo - see materials and methods), in cells treated as (E). Values normalized to Untreated siCtrl, set as 100%. Results are mean ±□SD of one of three representative assays (n=34) replicates. P values, compared to siCtrl were obtained by two-way ANOVA, Dunnett’s correction. G. KAP1-KZFPs recruit histone modifiers, ( e.g. SETDB1) inducing H3K9me3-mediated chromatin repression (top), often involving removal of active marks at enhancers (H3K4me1), activation sites (H3K27Ac), and promoters (H3K4me3) (bottom) (adapted from ). H. As (D), in U2OS siCtrl, siSETDB1 or siKAP1-depleted for 72 h; Results are mean□±□SEM; each dot represents one independent experiment of n□=□6; P values comparing to Ctrl obtained by two-way ANOVA, Dunnett’s correction. I. As (H), with siSETDB1 cells complemented with WT or catalytically inactive SETDB1 (Mut) for 24 h. J . As (A), in HAP-1 wild-type or knockout for KAP1. K. mRNA quantification probing different locations in Z DHHC20 transcripts (coding region: C1, C2; 5’ UTR: 1– 5 cover increasing lengths of 5’ ends.

Article Snippet: Plasmids expressing HA-tagged ZNF354A-HA, ZNF317-HA, ZNF677-HA, ZNF793-HA, KAP1-HA were obtained from the D. Trono lab . Plasmids expressing FLAG-ATF2 were from Origene (RC2118983), myc-SETDB1 (RC2266620) and plasmids expressing SP1-FLAG (25543) and FOXA1-V5 (153109) from Addgene.

Techniques: Western Blot, Control, Infection, Comparison, ChIP-sequencing, Activation Assay, Incubation, Expressing, Knock-Out

A . mRNA quantification measuring the efficiency of the ATF2, KAP1 and SETDB1 siRNA depletion in U2OS and HeLa cells. B-F. WB of ZDHHC20, Short-S and 20L isoforms, total and phospho-ATF2 in lysates from: B. HeLa cells over-expressing or not ATF2-HA (24 h), left untreated or treated with 10□ng/ml of proaerolysin (toxin) at 37□°C, washed, and incubated at 37□°C for 8 C. primary human airway epithelia extracts depicted in . D. Different mouse tissue extracts. E. Colon extracts from mice treated or not with DSS for 7 days followed by 3 days recovery. D-E with total and phosphor-KAP1. F. U2OS cells control (-) or overexpressing the indicated versions of ATF2 (WT, or phosphor mutants 6A, 6D, 4A, 4D, 2A, 2D). G. U2OS cells as F treated (24□h) with 100□nM JQ1 (FOXA1 inhibitor) or 100□nM mithramycin (SP1 inhibitor) or siRNA depleted for FOX1 and SP1 (72h). H . U2OS cells over-expressing (24 h) ATF2-HA, SP1-FLAG and FOXA1-V5, alone or in different combinations.

Journal: bioRxiv

Article Title: Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

doi: 10.1101/2025.02.25.640160

Figure Lengend Snippet: A . mRNA quantification measuring the efficiency of the ATF2, KAP1 and SETDB1 siRNA depletion in U2OS and HeLa cells. B-F. WB of ZDHHC20, Short-S and 20L isoforms, total and phospho-ATF2 in lysates from: B. HeLa cells over-expressing or not ATF2-HA (24 h), left untreated or treated with 10□ng/ml of proaerolysin (toxin) at 37□°C, washed, and incubated at 37□°C for 8 C. primary human airway epithelia extracts depicted in . D. Different mouse tissue extracts. E. Colon extracts from mice treated or not with DSS for 7 days followed by 3 days recovery. D-E with total and phosphor-KAP1. F. U2OS cells control (-) or overexpressing the indicated versions of ATF2 (WT, or phosphor mutants 6A, 6D, 4A, 4D, 2A, 2D). G. U2OS cells as F treated (24□h) with 100□nM JQ1 (FOXA1 inhibitor) or 100□nM mithramycin (SP1 inhibitor) or siRNA depleted for FOX1 and SP1 (72h). H . U2OS cells over-expressing (24 h) ATF2-HA, SP1-FLAG and FOXA1-V5, alone or in different combinations.

Techniques: Expressing, Incubation, Control

$A. Overview of the ZDHHC20 locus (first intron, exon, and 5’UTR - version hg19), Tracks indicate: in-frame transcription start codons (ATGs); transcript (GENCODE-V4 0lift37); SETDB1/KAP1,ATF2, SP1 and FOXA1 ChIP-seq (ENCODE) peaks; ZNF354A and other KZFP ChIP-seq peaks (squish view) from HEK293T cells obtained from KRABopedia ( https://tronoapps.epfl.ch/web/krabopedia/ ) (see also ). B. WB of ZDHHC20, Short-S and 20L isoforms and GAPDH loading control in U2OS cells depleted for SETDB1, KAP1, and the indicated KZFPs. C. Immunop□recipitation (IP) of endogenous KAP1 from U2OS cells over-expressing the indicated HA-tagged KZFPs (ZNF-HA) left untreated or treated 1 h with 10□ng/ml of proaerolysin (toxin) at 37□°C, washed, and incubated at 37□°C for 8 h. Blots show IP-fractions with Co-IP KZFPs and total cell extracts (TCE). D. As (C) in siCtrl or siATF2 (72 h) cells overexpressing ZNF354A-FLAG complemented with the indicated ATF2 constructs (WT, 6D or 6A mutants – 24 h). Results are mean□±□SEM; each dot represents one independent experiment (n□=□3); P values comparing to untreated (-) obtained by two-way ANOVA, Tukey’s correction. E. As (B) in human epithelial lung cells infected with SARS-CoV-2 (MOI 0.1). F. IP-KAP1, (as D), in U2OS cells siKAP1 (72 h) overexpressing WT or S473A KAP1 mutant treated with aerolysin (as C). Blots show KAP1 and correspondent anti-Ser- and Thr- phosphorylated protein bands (α-pSer/Thr) present in IP-KAP1 fractions G. As (B) in siKAP (72) U2OS cells expressing KAP1 WT, S473A-S/A, or S473D-S/D H . IP of ZNF354A from Calu-3 cells infected with SARS-CoV-2 (24 h), U2OS cells aerolysin-treated (as C). or U2OS siCtrl or siZNF354A depleted (as C). Blots show ZNF354A and α-pSer/Thr bands in IP-ZNF35A fractions and ZNF354A in TCE. I. IP-ZNF-HA in cells expressing the indicated ZNF-FLAG constructs and the indicated ATF2 constructs treated with aerolysin (as C). Blots show KAP1, ATF2, and α-pSer/Thr for IP-ZNF-HA fractions, and phosphor-KAP1 and total KAP1 and ATF2-HA for TCE. K. Schematic representation of the ATF2-KAP1-ZNF354A transcription regulation before and after stress conditions.$

Journal: bioRxiv

Article Title: Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

doi: 10.1101/2025.02.25.640160

Figure Lengend Snippet: A. Overview of the ZDHHC20 locus (first intron, exon, and 5’UTR - version hg19), Tracks indicate: in-frame transcription start codons (ATGs); transcript (GENCODE-V4 0lift37); SETDB1/KAP1,ATF2, SP1 and FOXA1 ChIP-seq (ENCODE) peaks; ZNF354A and other KZFP ChIP-seq peaks (squish view) from HEK293T cells obtained from KRABopedia ( https://tronoapps.epfl.ch/web/krabopedia/ ) (see also ). B. WB of ZDHHC20, Short-S and 20L isoforms and GAPDH loading control in U2OS cells depleted for SETDB1, KAP1, and the indicated KZFPs. C. Immunop□recipitation (IP) of endogenous KAP1 from U2OS cells over-expressing the indicated HA-tagged KZFPs (ZNF-HA) left untreated or treated 1 h with 10□ng/ml of proaerolysin (toxin) at 37□°C, washed, and incubated at 37□°C for 8 h. Blots show IP-fractions with Co-IP KZFPs and total cell extracts (TCE). D. As (C) in siCtrl or siATF2 (72 h) cells overexpressing ZNF354A-FLAG complemented with the indicated ATF2 constructs (WT, 6D or 6A mutants – 24 h). Results are mean□±□SEM; each dot represents one independent experiment (n□=□3); P values comparing to untreated (-) obtained by two-way ANOVA, Tukey’s correction. E. As (B) in human epithelial lung cells infected with SARS-CoV-2 (MOI 0.1). F. IP-KAP1, (as D), in U2OS cells siKAP1 (72 h) overexpressing WT or S473A KAP1 mutant treated with aerolysin (as C). Blots show KAP1 and correspondent anti-Ser- and Thr- phosphorylated protein bands (α-pSer/Thr) present in IP-KAP1 fractions G. As (B) in siKAP (72) U2OS cells expressing KAP1 WT, S473A-S/A, or S473D-S/D H . IP of ZNF354A from Calu-3 cells infected with SARS-CoV-2 (24 h), U2OS cells aerolysin-treated (as C). or U2OS siCtrl or siZNF354A depleted (as C). Blots show ZNF354A and α-pSer/Thr bands in IP-ZNF35A fractions and ZNF354A in TCE. I. IP-ZNF-HA in cells expressing the indicated ZNF-FLAG constructs and the indicated ATF2 constructs treated with aerolysin (as C). Blots show KAP1, ATF2, and α-pSer/Thr for IP-ZNF-HA fractions, and phosphor-KAP1 and total KAP1 and ATF2-HA for TCE. K. Schematic representation of the ATF2-KAP1-ZNF354A transcription regulation before and after stress conditions.

Techniques: ChIP-sequencing, Control, Expressing, Incubation, Co-Immunoprecipitation Assay, Construct, Infection, Mutagenesis

$Figure 1. MYH10, GOLGA4, and RAB3D form a triple complex in the Golgi, which is disbanded after EtOH treatment. (A) Immunostaining of Golgi (GOLGB1/Giantin, green) and myosin, heavy polypeptide 9, non-muscle (MYH9/MYH9-p-S1943) (red) in VA-13 cells treated with control or myosin, heavy polypeptide 10, non-muscle (MYH10/NMIIB) siRNAs. White squares indicate an area of Golgi membranes enlarged at the right; bars: 5 µm. (B) MYH10 W-B of the lysate samples from cells in A; ACTB/β-actin is a loading control. (C) Quantification of Pearson’s colocalization coefficient between GOLGB1 and MYH9-p-S1943 in cells from A; mean ± SD. (D) Immunostaining of TGOLN2 and MYH10 in control and EtOH-treated WIF-B cells and hepatocytes from mice fed with control or alcohol diet; bars: 2 µm for WIF-B cells, and 5 µm and 7 µm for hepatocytes from control and EtOH-fed mice, respectively. (E) Quantification of TGOLN2 and MYH10 colocalization in cells from D; mean ± SD. (F) RAB3D W-B of the postnuclear supernatant (PNS) isolated from control and EtOH-treated VA-13 cells. (G) Quantification of RAB3D densitometry from the samples in F counted from three independent experiments; unpaired t test. (H) RAB3D W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (I) Quantification of RAB3D densitometry from the samples in H counted from five independent experiments; one-way ANOVA test. (J, K) MYH9, MYH9-p-S1943 and RAB3D (J); MYH10 and GOLGA4 (K) W-B of the Golgi membrane fractions isolated from control and EtOH-treated VA-13 cells. Samples were normalized by TGOLN2. (L) Quantification of densitometry for the indicated proteins from the samples in J and K counted from three$

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 1. MYH10, GOLGA4, and RAB3D form a triple complex in the Golgi, which is disbanded after EtOH treatment. (A) Immunostaining of Golgi (GOLGB1/Giantin, green) and myosin, heavy polypeptide 9, non-muscle (MYH9/MYH9-p-S1943) (red) in VA-13 cells treated with control or myosin, heavy polypeptide 10, non-muscle (MYH10/NMIIB) siRNAs. White squares indicate an area of Golgi membranes enlarged at the right; bars: 5 µm. (B) MYH10 W-B of the lysate samples from cells in A; ACTB/β-actin is a loading control. (C) Quantification of Pearson’s colocalization coefficient between GOLGB1 and MYH9-p-S1943 in cells from A; mean ± SD. (D) Immunostaining of TGOLN2 and MYH10 in control and EtOH-treated WIF-B cells and hepatocytes from mice fed with control or alcohol diet; bars: 2 µm for WIF-B cells, and 5 µm and 7 µm for hepatocytes from control and EtOH-fed mice, respectively. (E) Quantification of TGOLN2 and MYH10 colocalization in cells from D; mean ± SD. (F) RAB3D W-B of the postnuclear supernatant (PNS) isolated from control and EtOH-treated VA-13 cells. (G) Quantification of RAB3D densitometry from the samples in F counted from three independent experiments; unpaired t test. (H) RAB3D W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (I) Quantification of RAB3D densitometry from the samples in H counted from five independent experiments; one-way ANOVA test. (J, K) MYH9, MYH9-p-S1943 and RAB3D (J); MYH10 and GOLGA4 (K) W-B of the Golgi membrane fractions isolated from control and EtOH-treated VA-13 cells. Samples were normalized by TGOLN2. (L) Quantification of densitometry for the indicated proteins from the samples in J and K counted from three

Article Snippet: RAB3D plasmid (full-length human RAB3D cDNA in pET28aLIC vector; Addgene, 25543; http://n2t.net/addgene:25543; RRID:Addgene 25543; deposited by Dr. Cheryl Arrowsmith Lab) and empty pET28a-LIC vector (26094) was obtained from DNASU Plasmid Repository.

Techniques: Immunostaining, Control, Isolation, Membrane

$Figure 2. Activation of autophagy flux in different cellular and animal models of chronic alcohol treatment. (A, B) MYH10 (A), MYH9, and WIPI2 (B) W-B of the liver tissue lysate samples obtained from the normal donors (N) and patients with alcoholic cirrhosis (AC); ACTB is a loading control. (C) Quantification of densitometry for the indicated proteins from the samples in A and B. (D) LC3B W-B of the lysates from hepatocytes of control and EtOH-fed rats. (E) Quantification of LC3B densitometry for the samples in D counted from five rat hepatocytes samples per group; unpaired t test. (F) LC3B W-B of the lysates from control and EtOH-treated VA-13 cells and co- treated with 25 µM HCQ. (G) Quantification of LC3B densitometry for the samples in F counted from three independent experiments; one-way ANOVA test. (H) WIPI2 W-B of the PNS (top panel) and Golgi membrane fraction (low panel) isolated from control and EtOH-treated VA-13 cells. (I) Quantification of WIPI2 densitometry for the Golgi samples in H counted from three independent experiments; unpaired t test. (J) WIPI2 W-B of the PNS (left panel) and Golgi membrane fraction (right panel) isolated from hepatocytes of rats fed with control or alcohol diet; TGOLN2 is a loading control, HSPA/HSP70 is ER/cytoplasm marker. (K) Quantification of WIPI2 densitometry for the Golgi samples in J counted from five rat hepatocytes samples per group; unpaired t test. (L) Representative 3D SIM images of mice hepatocytes from WT (control and EtOH-fed) and rab3d KO mice stained for WIPI2 (green) and TGOLN2 (red); bars: 5 µm. Images were reconstructed by Imaris. (M) Quantification of Manders’ coefficient of colocalization between WIPI2 and TGOLN2; n=15 3D SIM images for each group; Kruskal-Wallis test. (N-P) Immunostaining of TGOLN2 and LC3B in control and EtOH-treated WIF-B (N) and VA-13 (O) cells, and hepatocytes from control and EtOH-fed rats (P); bars: 5 µm. (Q) Quantification of Manders’ coefficient of colocalization between TGOLN2 and LC3B in cells from N-P; Mann-Whitney test. All data presented are representative of at least three independent experiments. For all graphs: ****p<0.0001, ***p<0.001,**p<0.01, and *p<0.05; mean ± SD.$

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 2. Activation of autophagy flux in different cellular and animal models of chronic alcohol treatment. (A, B) MYH10 (A), MYH9, and WIPI2 (B) W-B of the liver tissue lysate samples obtained from the normal donors (N) and patients with alcoholic cirrhosis (AC); ACTB is a loading control. (C) Quantification of densitometry for the indicated proteins from the samples in A and B. (D) LC3B W-B of the lysates from hepatocytes of control and EtOH-fed rats. (E) Quantification of LC3B densitometry for the samples in D counted from five rat hepatocytes samples per group; unpaired t test. (F) LC3B W-B of the lysates from control and EtOH-treated VA-13 cells and co- treated with 25 µM HCQ. (G) Quantification of LC3B densitometry for the samples in F counted from three independent experiments; one-way ANOVA test. (H) WIPI2 W-B of the PNS (top panel) and Golgi membrane fraction (low panel) isolated from control and EtOH-treated VA-13 cells. (I) Quantification of WIPI2 densitometry for the Golgi samples in H counted from three independent experiments; unpaired t test. (J) WIPI2 W-B of the PNS (left panel) and Golgi membrane fraction (right panel) isolated from hepatocytes of rats fed with control or alcohol diet; TGOLN2 is a loading control, HSPA/HSP70 is ER/cytoplasm marker. (K) Quantification of WIPI2 densitometry for the Golgi samples in J counted from five rat hepatocytes samples per group; unpaired t test. (L) Representative 3D SIM images of mice hepatocytes from WT (control and EtOH-fed) and rab3d KO mice stained for WIPI2 (green) and TGOLN2 (red); bars: 5 µm. Images were reconstructed by Imaris. (M) Quantification of Manders’ coefficient of colocalization between WIPI2 and TGOLN2; n=15 3D SIM images for each group; Kruskal-Wallis test. (N-P) Immunostaining of TGOLN2 and LC3B in control and EtOH-treated WIF-B (N) and VA-13 (O) cells, and hepatocytes from control and EtOH-fed rats (P); bars: 5 µm. (Q) Quantification of Manders’ coefficient of colocalization between TGOLN2 and LC3B in cells from N-P; Mann-Whitney test. All data presented are representative of at least three independent experiments. For all graphs: ****p<0.0001, ***p<0.001,**p<0.01, and *p<0.05; mean ± SD.

Techniques: Activation Assay, Control, Membrane, Isolation, Marker, Staining, Immunostaining, MANN-WHITNEY

Figure 3. MYH9, but not MYH10, drives phagophore formation in EtOH-treated cells. (A) VA-13 cells were treated with EtOH or MYH10 and RAB3D siRNAs, followed by transfection with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. The Golgi membranes were stained using anti-GOLGA4 Ab (magenta). White squares indicate an area of puncta around the Golgi membranes enlarged at the right. Bar size, 5 µm. (B) The autophagic index was counted as the ratio of the areas of autolysosomes (red) to autophagosomes (overlapped red and green); Kruskal-Wallis test. (C) Quantification of Manders’ coefficient of colocalization for red puncta (only autolysosomes) and GOLGA4; one-way ANOVA test. (D) Representative immunogold EM images of control and EtOH-treated VA-13 cells stained for MYH9 followed by secondary Ab conjugated with 10-nm gold particles; bars: 500 nm, (G - Golgi). Arrowheads denote MYH9-

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 3. MYH9, but not MYH10, drives phagophore formation in EtOH-treated cells. (A) VA-13 cells were treated with EtOH or MYH10 and RAB3D siRNAs, followed by transfection with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. The Golgi membranes were stained using anti-GOLGA4 Ab (magenta). White squares indicate an area of puncta around the Golgi membranes enlarged at the right. Bar size, 5 µm. (B) The autophagic index was counted as the ratio of the areas of autolysosomes (red) to autophagosomes (overlapped red and green); Kruskal-Wallis test. (C) Quantification of Manders’ coefficient of colocalization for red puncta (only autolysosomes) and GOLGA4; one-way ANOVA test. (D) Representative immunogold EM images of control and EtOH-treated VA-13 cells stained for MYH9 followed by secondary Ab conjugated with 10-nm gold particles; bars: 500 nm, (G - Golgi). Arrowheads denote MYH9-

Techniques: Transfection, Staining, Control

Figure 4. Evaluation of autophagy markers in different models of chronic alcohol administration. (A-D) LC3B (A) and SQSTM1 (C) W-B of the lysates from VA-13 cells: control and RAB3D KD; ACTB is a loading control. Quantification of RAB3D densitometry from the samples in A (B) and in C (D) counted from three independent experiments; unpaired t test. (E) LC3B W-B of the hepatocyte lysates from the WT mice fed with control or alcohol diet with or without HCQ treatment and rab3d KO mice receiving control diet only. (F, G) Quantification of LC3B-II (F) and LC3B-I (G) densitometry from the samples in E counted from three independent experiments; one-way ANOVA test. (H) SQSTM1 W-B of the hepatocyte lysates from the same group of mice in E. (I) Quantification of SQSTM1 densitometry from the samples in H counted from three independent experiments; one-way ANOVA test. (J) LC3B W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (K) Quantification of LC3B-II densitometry from the samples in J counted from three independent experiments; one-way ANOVA test. (L) WIPI2 W-B of the hepatocyte lysates from the same group of mice in E. (M) Quantification of WIPI2 densitometry from the samples in L counted from three independent experiments; one-way ANOVA test. (N) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with HCQ. (O) Quantification of LC3B-II densitometry from the samples in N counted from three independent experiments; one-way ANOVA test. (P) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with bafilomycin A1 (BAF). (Q) Quantification of LC3B-II densitometry from the samples in P counted from three independent experiments; one-way ANOVA test.

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 4. Evaluation of autophagy markers in different models of chronic alcohol administration. (A-D) LC3B (A) and SQSTM1 (C) W-B of the lysates from VA-13 cells: control and RAB3D KD; ACTB is a loading control. Quantification of RAB3D densitometry from the samples in A (B) and in C (D) counted from three independent experiments; unpaired t test. (E) LC3B W-B of the hepatocyte lysates from the WT mice fed with control or alcohol diet with or without HCQ treatment and rab3d KO mice receiving control diet only. (F, G) Quantification of LC3B-II (F) and LC3B-I (G) densitometry from the samples in E counted from three independent experiments; one-way ANOVA test. (H) SQSTM1 W-B of the hepatocyte lysates from the same group of mice in E. (I) Quantification of SQSTM1 densitometry from the samples in H counted from three independent experiments; one-way ANOVA test. (J) LC3B W-B of the lysate from VA-13 cells exposed to EtOH (35 mM) or EtOH and 5 µM MG132. (K) Quantification of LC3B-II densitometry from the samples in J counted from three independent experiments; one-way ANOVA test. (L) WIPI2 W-B of the hepatocyte lysates from the same group of mice in E. (M) Quantification of WIPI2 densitometry from the samples in L counted from three independent experiments; one-way ANOVA test. (N) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with HCQ. (O) Quantification of LC3B-II densitometry from the samples in N counted from three independent experiments; one-way ANOVA test. (P) LC3B W-B of the lysates from VA-13 cells: control and RAB3D KD treated with bafilomycin A1 (BAF). (Q) Quantification of LC3B-II densitometry from the samples in P counted from three independent experiments; one-way ANOVA test.

Techniques: Control

Figure 5. Differential distribution of GOLGA4 using Abs raised against its N or C terminus. (A-C) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against N terminus) in hepatocytes from control, EtOH-fed, and rab3d KO mice (A); VA-13 cells – control, EtOH-treated, and RAB3D KD (B); and hepatocytes from control and EtOH-fed rats (C); bars: 10 µm. (D) Quantification of Manders’ colocalization coefficient between TGOLN2 and GOLGA4 for samples in A-C. Kruskal- Wallis test for VA-13 cells and mice hepatocytes; Mann-Whitney test for rat hepatocytes; n indicates the number of cells counted. (E) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against C terminus) in VA-13 cells – control, EtOH-treated, and RAB3D KD; bars: 10 µm. (F) Quantification of Manders’ coefficient of colocalization between TGOLN2 and GOLGA4 for cells in E; Kruskal-Wallis test; n indicates the number of cells counted. (G) GOLGA4 W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs; ACTB is a loading control. (H) LC3B W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs and co-treated with EtOH. (I) Quantification of LC3B densitometry for the samples in H; unpaired t test. (J-M) Representative pre-embedding immunogold EM images of LC3B (1 nm) and GOLGA4 (10 nm) in control (J) and EtOH-treated (K and M) VA-13 cells. Red squares in J and K indicate LC3B-positive Golgi area highlighted at the right and indicated by arrow. L: Quantification of the number of LC3B-positive immunogold spots per Golgi stacks; n is the number of images. Inset a in M displays LC3B- and GOLGA4-positive immunogold spots (indicated by arrows) in fragmented Golgi membranes. Inset b indicates a phagophore that contains both GOLGA4 (arrowhead) and

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 5. Differential distribution of GOLGA4 using Abs raised against its N or C terminus. (A-C) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against N terminus) in hepatocytes from control, EtOH-fed, and rab3d KO mice (A); VA-13 cells – control, EtOH-treated, and RAB3D KD (B); and hepatocytes from control and EtOH-fed rats (C); bars: 10 µm. (D) Quantification of Manders’ colocalization coefficient between TGOLN2 and GOLGA4 for samples in A-C. Kruskal- Wallis test for VA-13 cells and mice hepatocytes; Mann-Whitney test for rat hepatocytes; n indicates the number of cells counted. (E) Immunostaining of TGOLN2 and GOLGA4 (Ab raised against C terminus) in VA-13 cells – control, EtOH-treated, and RAB3D KD; bars: 10 µm. (F) Quantification of Manders’ coefficient of colocalization between TGOLN2 and GOLGA4 for cells in E; Kruskal-Wallis test; n indicates the number of cells counted. (G) GOLGA4 W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs; ACTB is a loading control. (H) LC3B W-B of the lysate samples from VA-13 cells treated with control or GOLGA4 siRNAs and co-treated with EtOH. (I) Quantification of LC3B densitometry for the samples in H; unpaired t test. (J-M) Representative pre-embedding immunogold EM images of LC3B (1 nm) and GOLGA4 (10 nm) in control (J) and EtOH-treated (K and M) VA-13 cells. Red squares in J and K indicate LC3B-positive Golgi area highlighted at the right and indicated by arrow. L: Quantification of the number of LC3B-positive immunogold spots per Golgi stacks; n is the number of images. Inset a in M displays LC3B- and GOLGA4-positive immunogold spots (indicated by arrows) in fragmented Golgi membranes. Inset b indicates a phagophore that contains both GOLGA4 (arrowhead) and

Techniques: Immunostaining, Control, MANN-WHITNEY

$Figure 6. The conformational changes in GOLGA4 after RAB3D depletion. (A) The target epitopes of GOLGA4 Abs used. (B) Proximity ligation assay (PLA) of GOLGA4 central and C- terminal domains in control and VA-13 RAB3D KD cells. The proximity of GOLGA4's C terminus and the central region of its cytoplasmic domain was evaluated using Ms anti-GOLGA4 aa 2063- 2179 and Rb anti-GOLGA4 aa 1482-1584 Abs, accordingly. Red puncta indicate PLA signal, nucleus – blue, DAPI; bars: 5 μm. (C) Quantification of the number of PLA spots per nucleus from the samples in B. (D) The proximity of GOLGA4's C terminus and N terminus was evaluated in control and VA-13 RAB3D KD cells, using Ms anti-GOLGA4 aa 2063-2179 and Rb anti-GOLGA4 aa 1-150 Abs; bars: 5 μm. (E) Quantification of the number of PLA spots per nucleus from the samples in D. For graphs in C and E, dots indicate the average PLA signal from each image taken; n indicates the number of nuclei counted, Mann-Whitney test. (F) Left panel: GOLGA4 W- B of the Golgi fractions from control and RAB3D KD VA-13 cells. The samples were prepared under low (1%) concentration of β-mercaptoethanol and normalized by GOLGA4 monomer's bands. Given that depletion of RAB3D reduces the amount of GOLGA4, more proteins were loaded in the KD sample. GOLGA2 was used as a marker of Golgi membranes. Right panel: GOLGB1 (376 kDa) W-B of the control Golgi sample was used for the MW estimation. (G) Densitometry analysis of the ratio of the dimer:monomer and monomer:GOLGA2 from three independent experiment. (H) Schema illustrating a possible conformation of GOLGA4 dimer (monomers are shown in different colors for better visualization) in normal and fragmented Golgi$

Journal: Autophagy

Article Title: Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

doi: 10.1080/15548627.2024.2329476

Figure Lengend Snippet: Figure 6. The conformational changes in GOLGA4 after RAB3D depletion. (A) The target epitopes of GOLGA4 Abs used. (B) Proximity ligation assay (PLA) of GOLGA4 central and C- terminal domains in control and VA-13 RAB3D KD cells. The proximity of GOLGA4's C terminus and the central region of its cytoplasmic domain was evaluated using Ms anti-GOLGA4 aa 2063- 2179 and Rb anti-GOLGA4 aa 1482-1584 Abs, accordingly. Red puncta indicate PLA signal, nucleus – blue, DAPI; bars: 5 μm. (C) Quantification of the number of PLA spots per nucleus from the samples in B. (D) The proximity of GOLGA4's C terminus and N terminus was evaluated in control and VA-13 RAB3D KD cells, using Ms anti-GOLGA4 aa 2063-2179 and Rb anti-GOLGA4 aa 1-150 Abs; bars: 5 μm. (E) Quantification of the number of PLA spots per nucleus from the samples in D. For graphs in C and E, dots indicate the average PLA signal from each image taken; n indicates the number of nuclei counted, Mann-Whitney test. (F) Left panel: GOLGA4 W- B of the Golgi fractions from control and RAB3D KD VA-13 cells. The samples were prepared under low (1%) concentration of β-mercaptoethanol and normalized by GOLGA4 monomer's bands. Given that depletion of RAB3D reduces the amount of GOLGA4, more proteins were loaded in the KD sample. GOLGA2 was used as a marker of Golgi membranes. Right panel: GOLGB1 (376 kDa) W-B of the control Golgi sample was used for the MW estimation. (G) Densitometry analysis of the ratio of the dimer:monomer and monomer:GOLGA2 from three independent experiment. (H) Schema illustrating a possible conformation of GOLGA4 dimer (monomers are shown in different colors for better visualization) in normal and fragmented Golgi

Techniques: Proximity Ligation Assay, Control, MANN-WHITNEY, Concentration Assay, Marker

Fig. 2. Differential proteomic profiles of Olfm4-H and Olfm4-L neutrophil subsets in healthy blood donors. Olfm4-H and Olfm4- L neutrophils isolated from healthy blood donors (n = 8) were sort- ed by FACS and analysed by LC-MS/MS. a Volcano plot showing median log2 abundance ratios between the identified proteins in Olfm4-H and Olfm4-L neutrophils and their p values. b Left box- and-whisker plot showing proteins with a statistically significant positive median log2 abundance ratio, indicating increased abun- dance in the Olfm4-H neutrophils. Right box-and-whisker plot showing proteins with a statistically significant negative median log2 abundance ratio, indicating increased abundance in the Olfm4-L neutrophils. c Neutrophils from healthy blood donors (n = 7) were isolated and stained for proteins of interest and analysed by live imaging flow cytometry. Box-and-whisker plot comparing median fluorescence intensity of the immunofluorescently stained proteins Rab3d, S100A7, Rab3a, and CXCR1 between Olfm4-H and Olfm4-L neutrophils. Wilcoxon signed rank tests were used to test for statistical significance. Common contaminants and his- tones are not shown.

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 2. Differential proteomic profiles of Olfm4-H and Olfm4-L neutrophil subsets in healthy blood donors. Olfm4-H and Olfm4- L neutrophils isolated from healthy blood donors (n = 8) were sort- ed by FACS and analysed by LC-MS/MS. a Volcano plot showing median log2 abundance ratios between the identified proteins in Olfm4-H and Olfm4-L neutrophils and their p values. b Left box- and-whisker plot showing proteins with a statistically significant positive median log2 abundance ratio, indicating increased abun- dance in the Olfm4-H neutrophils. Right box-and-whisker plot showing proteins with a statistically significant negative median log2 abundance ratio, indicating increased abundance in the Olfm4-L neutrophils. c Neutrophils from healthy blood donors (n = 7) were isolated and stained for proteins of interest and analysed by live imaging flow cytometry. Box-and-whisker plot comparing median fluorescence intensity of the immunofluorescently stained proteins Rab3d, S100A7, Rab3a, and CXCR1 between Olfm4-H and Olfm4-L neutrophils. Wilcoxon signed rank tests were used to test for statistical significance. Common contaminants and his- tones are not shown.

Article Snippet: To quantify proteins of interest as identified by proteomics, AlexaFluor D ow nloaded from http://karger.com /jin/article-pdf/15/1/351/4124694/000527649.pdf by guest on 13 January 2025 Lundquist/Andersson/Chew/Das/ Turkina/Welin J Innate Immun 2023;15:351–364354 DOI: 10.1159/000527649 488-conjugated mouse anti-human Rab3d (sc-398727, Santa Cruz Biotechnology, Dallas, TX, USA) at 10 μg/mL, AlexaFluor 488-conjugated mouse anti-human S100A7 (NBP2-24911AF488, Novus Biologicals, Abingdon UK) at 31.5 μg/mL, AlexaFluor 488-conjugated mouse anti-human Rab3a (sc-365069, Santa Cruz Biotechnology) at 10 μg/mL or fluorescein isothiocyanate-conjugated mouse anti-human CXCR1 (320605, BioLegend) at 5 μg/mL were included in the primary antibody step.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Staining, Imaging, Flow Cytometry, Fluorescence

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