Journal: International Orthopaedics

Article Title: Abnormal expression of chondroitin sulphate N-acetylgalactosaminyltransferase 1 and Hapln-1 in cartilage with Kashin–Beck disease and primary osteoarthritis

doi: 10.1007/s00264-013-1937-y

Figure Lengend Snippet: Chondrocyte gene network of CSGalNAcT-1 was classified as glycan biosynthesis and metabolism and functional pathway

Article Snippet: For the primary antibody, polyclonal rabbit anti-human primary antibodies against CSGalNAcT-1 (1:50; ABGENT, San Diego, CA 92121, USA) and Hapln-1 (1:50; Abcam, UK) were used at 4 ° C overnight.

Techniques: Functional Assay

Journal: International Orthopaedics

Article Title: Abnormal expression of chondroitin sulphate N-acetylgalactosaminyltransferase 1 and Hapln-1 in cartilage with Kashin–Beck disease and primary osteoarthritis

doi: 10.1007/s00264-013-1937-y

Figure Lengend Snippet: CSGalNAcT-1 and Hapln-1 mRNA expression in osteoarthritis (OA) cartilage, Kashin-Beck disease (KBD) cartilage and normal cartilage. CSGalNAcT-1 and Hapln-1 mRNA levels reach statistical significance. * Denotes significant difference at P < 0.05

Article Snippet: For the primary antibody, polyclonal rabbit anti-human primary antibodies against CSGalNAcT-1 (1:50; ABGENT, San Diego, CA 92121, USA) and Hapln-1 (1:50; Abcam, UK) were used at 4 ° C overnight.

Techniques: Expressing

Journal: International Orthopaedics

Article Title: Abnormal expression of chondroitin sulphate N-acetylgalactosaminyltransferase 1 and Hapln-1 in cartilage with Kashin–Beck disease and primary osteoarthritis

doi: 10.1007/s00264-013-1937-y

Figure Lengend Snippet: Comparison of CSGalNAcT-1 expression in the cartilage from osteoarthritis (OA), Kashin-Beck disease (KBD) and normal groups. a , b , c Denote the upper of normal, OA and KBD cartilage, respectively. d , e , f Denote the middle of normal, OA and KBD cartilage, respectively. g , h , i Denote the deep of normal, OA and KBD cartilage, respectively

Article Snippet: For the primary antibody, polyclonal rabbit anti-human primary antibodies against CSGalNAcT-1 (1:50; ABGENT, San Diego, CA 92121, USA) and Hapln-1 (1:50; Abcam, UK) were used at 4 ° C overnight.

Techniques: Expressing

Journal: International Orthopaedics

Article Title: Abnormal expression of chondroitin sulphate N-acetylgalactosaminyltransferase 1 and Hapln-1 in cartilage with Kashin–Beck disease and primary osteoarthritis

doi: 10.1007/s00264-013-1937-y

Figure Lengend Snippet: CSGalNAcT-1 and Hapln-1 protein level expression reduced in osteoarthritis (OA) and Kashin-Beck disease (KBD) cartilage compared with normal controls. Wnt 3a, β-catenin and Runx-2 level expression increased in OA and KBD cartilage compared with normal controls. A KBD free-Se group. B 0.25 μg/ml Se + KBD group. C 0.10 μg/ml Se + KBD group. D 0.05 μg/ml Se + KBD group

Article Snippet: For the primary antibody, polyclonal rabbit anti-human primary antibodies against CSGalNAcT-1 (1:50; ABGENT, San Diego, CA 92121, USA) and Hapln-1 (1:50; Abcam, UK) were used at 4 ° C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: Jaw1 increases the Ca 2+ influx depending on its own expression level. ( A , B , E , F , L ) Mean curves of relative Fluo-4 ( A , E , L ) or Rhod-2 ( B , F ) intensity upon 100 µM ATP stimulation measured using a plate reader. WT, Jaw1 KO #7, and Jaw1 KO #17 cells ( A , B ); Jaw1 KO, Jaw1 IE, and Δcoil IE cells ( E , F ); or Jaw1 KO, Jaw1 IE medium, and Jaw1 IE high cells ( L ). The closed triangles indicate the time point of 100 µM ATP solution supplementation. ( C , D , G , H , M ) AUC in ( A ), ( B ), ( E ), ( F ), and ( L ) are shown in ( C ), ( D ), ( G ), ( H ), and ( M ), respectively. n = 3. ( I ) Relative expression level of jaw1 mRNA to gapdh in WT and Jaw1 IE cells measured by RT-qPCR. N = 3. ( J ) Jaw1 KO and Jaw1 IE cells treated with 0.25 or 200 ng/mL of Dox and subjected to western blotting. Images used for western blots are shown in Supplementary Fig. online. ( K ) The relative protein expression level of Jaw1 with α-Tubulin in ( J ). n = 3. The error bar shows ± S.D.; n.s., non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, Tukey–Kramer’s t -test.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: Jaw1 increases Ca 2+ influx by changing the Ca 2+ influx pattern. ( A , B , C ) Representative relative intensity traces upon 100 µM ATP stimulation: ( A ) single, ( B ) oscillation, and ( C ) steady reduction types. ( D , G ) Mean ( D ) or five representative ( G ) curves of the relative intensity in Jaw1 KO, Jaw1 IE, and Δcoil IE cells. The closed triangles indicate the time point of 100 µM ATP solution supplementation. n = 200. ( E , F ) AUC ( E ) and maximum amplitude ( F ) of the relative intensity from ( D ). ( H ) Ca 2+ influx type classification in Jaw1 KO, Jaw1 IE, and Δcoil cells. Four independent experiments (n = 50). The error bars show ± S.D.; n.s., non-significant; * p < 0.05; *** p < 0.001; **** p < 0.0001, Tukey–Kramer’s t -test.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques:

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: The Jaw1 augmentative effect on the Ca 2+ influx is maintained even under weak GPCR stimulation. ( A , E ) Five representative relative Fluo-4 fluorescence intensity responses to the stimulation with 1 µM ( A ) or 0.3 µM ( E ) ATP out of 200 cells. The closed triangles show the time point of ATP stimulation. ( B , F ) Ca 2+ influx type classification in each cell line upon stimulation with 1 µM ( B ) or 0.3 µM ( F ) ATP. The ratios were calculated based on the average of four independent wells. ( C , D , G , H ) Graph representing the AUC ( C , G ) and maximum amplitude ( D , H ) of the relative Fluo-4 fluorescence intensity in each cell line upon stimulation with 1 µM ( C , D ) or 0.3 µM ( G , H ) ATP in 200 cells. The error bar shows ± S.D.; **** p < 0.0001. Student’s t -test.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: Jaw1 accelerates Ca 2+ efflux from ER . ( A , D ) Mean ( A ) or five representative ( D ) curves of the relative intensity upon 100 µM ATP stimulation under extracellular Ca 2+ free condition in Jaw1 KO and Jaw1 IE cells. n = 200. The closed triangle shows the time point of 100 µM ATP solution supplementation. ( B , C ) AUC ( B ) or maximum amplitude ( C ) of the relative intensity from ( A ). ( E ) Mean curves of the relative intensity upon Ca 2+ depletion in the ER by 2 µM thapsigargin supplementation in 0 mM Ca 2+ and replenishment with 2 mM Ca 2+ . The closed triangle shows the time point of 2 µM thapsigargin solution supplementation. The area surrounded with a black box is enlarged in the right. n = 3. ( F , G ) Maximum amplitude in SOCE ( F ) and time duration to reach the maximum amplitude in the Ca 2+ leakage ( G ) from ( E ). The error bar shows ± S.D.; n.s., non-significant; * p < 0.05; **** p < 0.0001, Student’s t -test.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques:

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: Jaw1 increases the activity of all ITPR subtypes with subtle differences. ( A ) Jaw1 KO, Jaw1 IE, and Δcoil IE cell lysates were subjected to co-immunoprecipitation followed by western blotting. ( B ) Jaw1 KO, R1 SE #11 or #17, R2 SE #5 or #9, and R3 SE #5 or #7 cells were subjected to western blotting. Images used for western blots of ( A ) and ( B ) are shown in Supplementary Fig. , online. ( C , D , F , G , I , J ) Mean ( C , F , I ) or five representative ( D , G , J ) curves of the relative intensity in R1 SE, R2 SE, and R3 SE with or without Jaw1 IE cells. Closed triangles indicate the time point of 100 µM ATP solution supplementation. n = 200. ( E , H , K ) Classification of the Ca 2+ influx type in each cell line. Four independent experiments (n = 50). ( L , M ) AUC ( L ) or maximum amplitude ( M ) of the relative intensity from ( C ), ( F ), and ( I ). The error bar shows ± S.D.; n.s., non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Jaw1/LRMP increases Ca 2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

doi: 10.1038/s41598-022-13620-4

Figure Lengend Snippet: A model of the Jaw1 function as a regulator of the ITPRs activity upon GPCR stimulation. ( A ) Jaw1 promotes Ca 2+ release activity of ITPRs and increases Ca 2+ influx into cytoplasm and mitochondria upon GPCR stimulation. Jaw1 changes the Ca 2+ influx pattern from oscillated response to continuous response in high GPCR stimulation, and from weak response to strong response in low GPCR stimulation.

Article Snippet: Rabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech).

Techniques: Activity Assay

Journal: Journal of Investigative Medicine

Article Title: MiR-3150b inhibits hepatocellular carcinoma cell proliferation, migration and invasion by targeting GOLPH3

doi: 10.1136/jim-2019-001181

Figure Lengend Snippet: Expression of miR-3150b was downregulated in HCC cell lines. Relative mRNA expression of miR-3150b normal liver HL7702 cells and four GC cell lines with different differentiation status (MHCC-97L, HepG2, SMMC-7721 and SNU-398) determined by quantitative RT-PCR. All experiments were performed in triplicate with at least three independent experiments. Data were presented as the mean±SD. * p<0.05, ** p<0.01 vs HL7702 cells. GOLPH3, Golgi phosphoprotein 3; HCC, hepatocellular carcinoma.

Article Snippet: The blots were incubated with rabbit antihuman polyclonal antibodies against GOLPH3 (catalog no. 19 112–1-AP; 1:2000 dilution; Peprotech, Rocky Hill, New Jersey, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Investigative Medicine

Article Title: MiR-3150b inhibits hepatocellular carcinoma cell proliferation, migration and invasion by targeting GOLPH3

doi: 10.1136/jim-2019-001181

Figure Lengend Snippet: MiR-3150b suppressed HCC cell proliferation relative miR-3150b expression (A) and CCK-8 cell viability assay (B) in HepG2 cells with transfection of miR-3150b mimic or si-GOLPH3, and in SNU-398 cells transfected with miR-3150b inhibitor or pcDNA3.1-GOLPH3. All experiments were performed in triplicate with at least three independent experiments. Data were presented as the mean±SD. ** p<0.01 vs mimic NC or inhibitor NC group. CCK-8, Cell Counting Kit-8; GOLPH3, Golgi phosphoprotein 3; HCC, hepatocellular carcinoma; NC, negative control.

Article Snippet: The blots were incubated with rabbit antihuman polyclonal antibodies against GOLPH3 (catalog no. 19 112–1-AP; 1:2000 dilution; Peprotech, Rocky Hill, New Jersey, USA).

Techniques: Expressing, CCK-8 Assay, Viability Assay, Transfection, Cell Counting, Negative Control

Journal: Journal of Investigative Medicine

Article Title: MiR-3150b inhibits hepatocellular carcinoma cell proliferation, migration and invasion by targeting GOLPH3

doi: 10.1136/jim-2019-001181

Figure Lengend Snippet: GOLPH3 was a direct target of miR-3150b. (A) alignment of miR-3150b with GOLPH3 3'-UTR sequences. HEK293T cells were co-transfected with luciferase reporter with wild-type (WT) GOLPH3 3'-UTR or with mutant GOLPH3 3'-UTR, and miR-3150b or mimic NC for 48 hours. The relative luciferase activity was analyzed by luciferase assay. The mRNA (B) and protein (C) levels of GOLPH3 in HepG2 cells transfected with miR-3150b mimic or mimic NC, and SNU-398 cells transfected with miR-3150b inhibitor or inhibitor NC using quantitative RT-PCR and western blot analysis. All experiments were performed in triplicate with at least three independent experiments. Data were presented as the mean±SD. ** p<0.01, *** p<0.001 vs mimic NC or inhibitor NC group. GOLPH3, Golgi phosphoprotein 3; HCC, hepatocellular carcinoma; NC, negative control; UTR, untranslated region.

Article Snippet: The blots were incubated with rabbit antihuman polyclonal antibodies against GOLPH3 (catalog no. 19 112–1-AP; 1:2000 dilution; Peprotech, Rocky Hill, New Jersey, USA).

Techniques: Transfection, Luciferase, Mutagenesis, Activity Assay, Quantitative RT-PCR, Western Blot, Negative Control