mouse lamp-1/cd107a lumenal domain antibody  (Bio-Techne corporation)

Journal: Journal of Cell Science

Article Title: Syntaxin-2 balances phagocytic uptake and phagolysosomal clearance in macrophages

doi: 10.1242/jcs.263855

Figure Lengend Snippet: Stx2 exhibits multiple subcellular distributions in macrophages, and inhibits engagement and uptake of IgG-opsonized particles. (A–F) DIC and confocal images of endogenous Stx2 with endogenous (A) β-actin (ACTB), (B) RAB5A, (C) Rab11, (D) VAMP4, (E) VAMP7 and (F) LAMP1 at steady-state. (G) DIC and confocal images of RAW 264.7 macrophages fed with OPZ (white asterisks) for 1h and probed for endogenous Stx2 and LAMP1. Boxed areas enlarged in the insets. ‘ r ’ values show degree of colocalization. Scale bars: 10 μm (main images) and 5 μm (insets). DAPI denotes the nucleus. All images representative of three independent repeats. (H) Western blot of Stx2 and β-actin (ACTB, loading control) from RAW 264.7 macrophages (WT) and macrophages transduced with scrambled (sc) shRNA (control) or shRNAs targeted to Stx2 (Stx2-KD). (I) Quantified Stx2 band density normalized to ACTB (loading control). N =3 independent experiments. Results are mean±s.e.m. ns, not significant; * P <0.05. (J) DIC and stacked (three consecutive optical sections) confocal images from 15, 30 and 60 min OPZR-incubated macrophages also probed for Stx2. DAPI marks the nucleus. Scale bars: 10 μm. (K) Quantification of engaged OPZR per macrophage (15 min: control, n =61; Stx2-KD, n =82; 30 min: control, n =56; Stx2-KD, n =109; 60 min: control, n =111; Stx2-KD, n =86). N =3 independent experiments. Results are mean±s.e.m. **** P <0.0001. (L) Maximum intensity projection confocal images of inside-outside stained macrophages incubated with OPZR for 15, 30 and 60 min. Scale bars: 10 µm. (M,N) Quantification of (M) phagocytic index and (N) phagocytic rate for OPZR analyzed at 15 min (control, n =75; Stx2-KD, n =68), 30 min (control, n =273; Stx2-KD, n =344) and 60 min (control, n =165; Stx2-KD, n =190). N =3 independent experiments. M shown as violin plots with median and quartiles marked. For N, results are mean±s.e.m. ** P <0.01, **** P <0.0001. All statistical tests were two-tailed unpaired Student's t -tests. See also Fig. S1 .

Article Snippet: Primary antibodies diluted in primary antibody buffer [1% BSA (w/v) in 50 mM Tris-HCl pH 7.4)] were used to probe membranes for Stx2 (Thermo Fisher Scientific, catalog #PA5-87903, 1:1000 dilution), Rab5A (Cell Signaling Technology, catalog #46449; 1:1000 dilution), VAMP4 (Thermo Fisher Scientific, catalog #PA1-768, 1:1000 dilution), LAMP1 (R&D Systems, catalog #AF4320, 1:1000 dilution), Golgin160 (Santa Cruz Biotechnology, catalog #sc-374596, 1:500 dilution), VAMP7 (Thermo Fisher Scientific, catalog #PA5-116892, 1:1000 dilution), VAMP8 (Proteintech, catalog #15546-1-AP, 1:1000 dilution), cathepsin L (R&D Systems, catalog #1515, 1:1000 dilution), cathepsin B (Cell Signaling Technology, catalog #31718, 1:1000 dilution), cathepsin D (Proteintech, catalog #21327-1-AP, 1:1000 dilution), ATP6V1A (Cell Signaling Technology, catalog #39517, 1:1000 dilution), TFEB (Thermo Fisher Scientific, catalog #PA5-96632, 1:1000 dilution), histone H3 (Cell Signaling Technology, catalog #9715, 1:2000 dilution), LC3A/B (Cell Signaling Technology, catalog #12741, 1:400 dilution), SQSTM1/p62 (Cell Signaling Technology, catalog #39749, 1:800 dilution), β-actin (Cell Signaling Technology, catalog #3700, 1:1000 dilution) and tubulin (Cell Signaling Technology, catalog #3873; 1:1000 dilution) for 12 h at 4°C.

Techniques: Western Blot, Control, Transduction, shRNA, Incubation, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Syntaxin-2 balances phagocytic uptake and phagolysosomal clearance in macrophages

doi: 10.1242/jcs.263855

Figure Lengend Snippet: Increased lysosome content couples with diminished pro-cathepsin secretion in Stx2-depleted macrophages. (A) DIC and stacked (three consecutive optical sections) confocal images of LAMP1 immunostained macrophages. Boxed areas enlarged in inset. Nuclei labeled with DAPI. Scale bars: 10 µm (main images) and 2 µm (inset). (B) Macrophages quantified for number (left) and size (right) of LAMP1 puncta from control ( n =54) and Stx2-KD ( n =54) cells. N =3 independent experiments. Results are mean±s.e.m. (left) and violin plots with median and quartiles marked (right). ** P <0.01; *** P <0.001. Analyses of macrophage LAMP1 puncta in C5 and C6 macrophages are in Fig. S5C–E . (C) DIC and confocal merged images for CTSL, CTSB and CTSD. Boxes are magnified and split into separate channels in inset. Scale bars: 10 µm (main images) and 2 µm (inset). (D) Quantified (control, n =73; Stx2-KD, n =56) fluorescence intensity for cathepsins. N =3 independent experiments. Results are mean±s.e.m. * P <0.05, *** P <0.001. (E) Western blots of macrophage total cell lysates (TCL) for CTSL, CTSB, CTSD and ACTB (loading control). (F) Quantified band density for pro- and mature forms of CTSL, CTSB and CTSD, normalized to ACTB. N =3 independent experiments. Results are mean±s.e.m. * P <0.05, ** P <0.01, **** P <0.0001. (G) Western blot detection for TFEB in nuclear (Nu) and cytosolic (Cyto) fractions. ACTB was used as a marker as well as a loading control for TCL and Cyto fraction. Histone served as a marker and loading control for Nu fractions. (H) TFEB band density normalized to ACTB for TCL and Cyto fractions and to histone for Nu fractions. N =3 independent experiments. Results are mean±s.e.m. ns, not significant. (I) Western blot detection of macrophage secreted pro- and mature CTSL and CTSB. Tubulin and Ponceau S-stained membranes were used as loading control for TCL and media, respectively. (J,K) Quantified secreted (J) pro-CTSL (left) and pro-CTSB (right) and (K) mature CTSL (left) and mature CTSB (right). TCL bands were normalized to tubulin and media bands were normalized to Ponceau S-stained lanes. N =3 independent experiments. Results are mean±s.e.m. ns, not significant. ** P <0.01. All statistical tests were two-tailed unpaired Student's t -tests. See also Fig. S5 .

Article Snippet: Primary antibodies diluted in primary antibody buffer [1% BSA (w/v) in 50 mM Tris-HCl pH 7.4)] were used to probe membranes for Stx2 (Thermo Fisher Scientific, catalog #PA5-87903, 1:1000 dilution), Rab5A (Cell Signaling Technology, catalog #46449; 1:1000 dilution), VAMP4 (Thermo Fisher Scientific, catalog #PA1-768, 1:1000 dilution), LAMP1 (R&D Systems, catalog #AF4320, 1:1000 dilution), Golgin160 (Santa Cruz Biotechnology, catalog #sc-374596, 1:500 dilution), VAMP7 (Thermo Fisher Scientific, catalog #PA5-116892, 1:1000 dilution), VAMP8 (Proteintech, catalog #15546-1-AP, 1:1000 dilution), cathepsin L (R&D Systems, catalog #1515, 1:1000 dilution), cathepsin B (Cell Signaling Technology, catalog #31718, 1:1000 dilution), cathepsin D (Proteintech, catalog #21327-1-AP, 1:1000 dilution), ATP6V1A (Cell Signaling Technology, catalog #39517, 1:1000 dilution), TFEB (Thermo Fisher Scientific, catalog #PA5-96632, 1:1000 dilution), histone H3 (Cell Signaling Technology, catalog #9715, 1:2000 dilution), LC3A/B (Cell Signaling Technology, catalog #12741, 1:400 dilution), SQSTM1/p62 (Cell Signaling Technology, catalog #39749, 1:800 dilution), β-actin (Cell Signaling Technology, catalog #3700, 1:1000 dilution) and tubulin (Cell Signaling Technology, catalog #3873; 1:1000 dilution) for 12 h at 4°C.

Techniques: Labeling, Control, Fluorescence, Western Blot, Marker, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Syntaxin-2 balances phagocytic uptake and phagolysosomal clearance in macrophages

doi: 10.1242/jcs.263855

Figure Lengend Snippet: Stx2 is required for macrophage phagosome maturation and acidification. (A) DIC and confocal images of OPZ-fed macrophages immunostained for LAMP1. White asterisks show the OPZ-containing phagosomes. Boxed regions magnified in the inset. DAPI marks the nuclei. Scale bars: 10 µm (main images) and 2 μm (inset). (B) Quantification (15 min: control, n =35; Stx2-KD, n =42; 30 min: control, n =29; Stx2-KD, n =68; 60 min: control, n =29; Stx2-KD, n =53) of the phagosomal LAMP1 fluorescence intensity. N =3 independent experiments. Results are mean±s.e.m. **** P <0.0001. (C) DIC and confocal images of 60 min OPZ-fed macrophages immunostained for CTSL, CTSB and CTSD. Boxed areas are magnified and shown as separate channels in inset. DAPI labeled the nuclei. Scale bars: 10 µm (main images) and 2 μm (inset). (D) Quantification (control, n =46; Stx2-KD, n =43) of the phagosomal fluorescence intensity for CTSL, CTSB and CTSD. N =3 independent experiments. Results are mean±s.e.m. * P <0.05, **** P <0.0001. (E) Western blots of macrophage total cell lysates (TCL) and purified phagosomes (phago) probed for LAMP1, CTSL, CTSB, CTSD, ATP6V1A and tubulin (loading control for TCL and negative control for ‘phago’). Ponceau S-stained membrane shows uniform loading. (F–J) Quantified band density of (F) LAMP1, (G) CTSL, (H) CTSB, (I) CTSD and (J) ATP6V1A, normalized to tubulin (for TCL) or Ponceau S-stained lanes for phagosomes. N ≥3 independent experiments. Results are mean±s.e.m. ** P <0.01; *** P <0.001; **** P <0.0001. (K) DIC and confocal images of 30 and 60 min OpHRZ-fed macrophages. DAPI used to visualize nuclei. Scale bars: 10 µm. (L) Quantification of fluorescence intensity (higher intensity indicates lower pH) of internalized (red) OpHRZ particle (30 min: control, n =113; Stx2-KD, n =66; 60 min: control, n =129; Stx2-KD, n =131). N =3 independent experiments. Results are mean±s.e.m. **** P <0.0001. All statistical tests were two-tailed unpaired Student's t -tests.

Article Snippet: Primary antibodies diluted in primary antibody buffer [1% BSA (w/v) in 50 mM Tris-HCl pH 7.4)] were used to probe membranes for Stx2 (Thermo Fisher Scientific, catalog #PA5-87903, 1:1000 dilution), Rab5A (Cell Signaling Technology, catalog #46449; 1:1000 dilution), VAMP4 (Thermo Fisher Scientific, catalog #PA1-768, 1:1000 dilution), LAMP1 (R&D Systems, catalog #AF4320, 1:1000 dilution), Golgin160 (Santa Cruz Biotechnology, catalog #sc-374596, 1:500 dilution), VAMP7 (Thermo Fisher Scientific, catalog #PA5-116892, 1:1000 dilution), VAMP8 (Proteintech, catalog #15546-1-AP, 1:1000 dilution), cathepsin L (R&D Systems, catalog #1515, 1:1000 dilution), cathepsin B (Cell Signaling Technology, catalog #31718, 1:1000 dilution), cathepsin D (Proteintech, catalog #21327-1-AP, 1:1000 dilution), ATP6V1A (Cell Signaling Technology, catalog #39517, 1:1000 dilution), TFEB (Thermo Fisher Scientific, catalog #PA5-96632, 1:1000 dilution), histone H3 (Cell Signaling Technology, catalog #9715, 1:2000 dilution), LC3A/B (Cell Signaling Technology, catalog #12741, 1:400 dilution), SQSTM1/p62 (Cell Signaling Technology, catalog #39749, 1:800 dilution), β-actin (Cell Signaling Technology, catalog #3700, 1:1000 dilution) and tubulin (Cell Signaling Technology, catalog #3873; 1:1000 dilution) for 12 h at 4°C.

Techniques: Control, Fluorescence, Labeling, Western Blot, Purification, Negative Control, Staining, Membrane, Two Tailed Test